1. Prepare the reaction mixture at room temperature in the order indicated:

   Component	Volume
   	Plasmid DNA	Unpurified PCR product	Genomic DNA
   Water, nuclease-free*	15 µl	17 µl	30 µl
   10X FastDigest® buffer or 10X FastDigest® Green buffer	2 µl	2 µl**	5 µl
   DNA*	2 µl (up to 1 µg)	10 µl (~0.2 µg)	10 µl (5 µg)
   FastDigest® enzyme	1 µl	1 µl	5 µl
   Total volume	20 µl	30 µl	50 µl

2. Mix gently and spin down.
3. Incubate at 37°C in a heat block or water thermostat for 5 min.***
4. Inactivate the enzyme (optional).***

Note

1. Pipette 2 µl of each DNA sample* into labeled tubes.
2. Prepare a master mix for n+1 samples.
   Example of master mix (for 10 samples of plasmid DNA):

   Water, nuclease-free	(10 + 1) x 15 µl = 165 µl
   10X FastDigest® buffer or 10X FastDigest® Green buffer	(10 + 1) x 2 µl = 22 µl
   FastDigest® enzyme	(10 + 1) x 1 µl = 11 µl

3. Add 18 µl of master mix into tubes containing DNA

Note

• Use 1 µl of each enzyme and scale up the reaction conditions appropriately.
• The combined volume of all added enzymes should not exceed 1/10 of the total reaction volume.
• If enzymes require different incubation temperature, perform sequential DNA cleavage: complete the first digestion reaction at the lower temperature, add the scond enzyme and increase the digestion temperature for the second enzyme cleavage.

1. Add the following reaction components in the order indicated:

   Water, nuclease-free	16-16.5 µl
   10X recommended buffer for restriction enzyme	2 µl
   Substrate DNA	1 µl (~1 µg)
   Restriction enzyme	0.5-1 µl (5-10 u)
   Total volume	20 µl

2. Mix gently and spin down briefly.
3. Incubate at the optimal reaction temperature for 1-16 hours.

Note

• The digestion reaction may be scaled either up or down.
• Some enzymes require additional components to obtain the stated activity. In these cases, add the required additive and adjust the volume of water appropriately.

1. Add the following reaction components in the order indicated:

   PCR reaction mixture	10 µl (~0.1-0.5 µg of DNA)
   Water, nuclease-free	16-17 µl
   10X recommended buffer for restriction enzyme	2 µl*
   Restriction enzyme	1-2 µl (10-20 u)
   Total volume	30 µl

   * Only 2 µl of 10X reaction buffer is required for unpurified PCR product in a 30 µl reaction volume.
2. Mix gently and spin down briefly.
3. Incubate at the optimal reaction temperature for 1-16 hours.

Note

• For cloning applications, purification of PCR products prior to digestion is necessary to remove the active thermophilic DNA polymerase present in the PCR mixture. DNA polymerases may alter the ends of the cleaved DNA and reduce the yield of ligation.
• If the restriction enzyme requires special additives (e.g., SAM), reduce the amount of water appropriately.
• If cleavage of the PCR product is inefficient purify the PCR products with the GeneJET™ PCR Purification Kit (#K0702) prior to digestion.
• After digestion, gel-purify the PCR product with the GeneJET™ Gel Extraction Kit (#K0692) or Silica Bead DNA Gel Extraction Kit (#K0513) to remove short DNA fragments which compete with the insert in a ligation reaction.

1. Embed the substrate DNA in 1% low melting temperature agarose, 1 µg / 30 µl.
2. Prepare ~30 µl agarose plugs with a GelSyringe® or similar agarose dispensing system.
3. Equilibrate the plug in 100 µl of the appropriate 1X restriction enzyme buffer for 15 min.
4. Place the plug in 100 µl of fresh 1X buffer containing the restriction enzyme (see Table "Digestion of Agarose-embedded DNA").
5. Incubate at 30-37°C for mesophilic enzymes or at 50-55°C for thermophilic enzymes for 4-16 hours.

1. Immerse an agarose plug in 50-100 µl of the 1X Tango™ buffer without Mg-acetate* (supplied with the enzyme). The volume of the buffer should be sufficient to completely cover the plug.
2. Add 20 u of the enzyme.
3. Incubate 2 hours on ice.
4. Add 1/10 volume of the 100 mM Mg-acetate solution (supplied with the enzyme).
5. Incubate at 37°C for 1 hour.

Note

• suboptimal concentration of the restriction enzyme in the reaction mixture;
• short incubation time;
• incubation at a suboptimal temperature.

• Prepare your DNA sample, set up the PCR mixture, perform thermal cycling and analyze PCR products in separate areas.
• Set up mixtures for PCR in a laminar flow cabinet equipped with an UV lamp.
• Wear fresh gloves for DNA purification and reaction set up.
• Use containers dedicated for PCR. Use positive displacement pipettes, or use pipette tips with aerosol filters to prepare DNA samples and set up PCR.
• Use certified reagents, including high quality water (e.g., Water, nuclease-free).
• Always perform No-Template-Control (NTC) reactions to check for the absence of contamination.
  For detailed instructions for the set-up of a PCR laboratory and its maintenance, refer to PCR Methods and Applications, 3, 2, S1-S14, 1993.

• PCR primers are generally 15-30 nucleotides long.
• Optimal GC content of the primer is 40-60%. Ideally, C and G nucleotides should be distributed uniformly along the primer.
• Prefer one or two G or C at the 3'-end of the primer, but avoid placing more than three G or C nucleotides at the 3'-end to lower the risk of nonspecific priming.
• Avoid primer self-complementarities, complementarities between the primers and direct repeats in a primer to prevent hairpin formation and primer dimerization.
• Check for possible complementary sites between primers and template DNA.
• When designing degenerate primers, place at least 3 conservative nucleotides at the 3'-end.
• Differences in melting temperatures (Tm) of the two primers should not exceed 5°C for conventional PCR.

• For primers containing less than 25 nucleotides, the approx. melting temperature (Tm) can be calculated using the following equation:
  Tm = 4 (G + C) + 2 (A + T), where G, C, A, T – number of respective nucleotides in the primer.
• If the primer contains more than 25 nucleotides specialized computer programs e.g, REviewer™ are recommended to account for interactions of adjacent bases, effect of salt concentration, etc.
• For calculation of primer melting temperature only consider nucleotides homologous to the template.

• When introducing restriction endonuclease sites into primers for subsequent digestion and cloning of the PCR product, refer to tables "Cleavage Efficiency Close to the Termini of PCR Fragments" or "Reaction Conditions for FastDigest® Restriction Enzymes" to determine the number of extra bases outside of the recognition sequence that are required for efficient cleavage.

• Taq DNA Polymerase at Fermentas is supplied with two buffers: Taq buffer with KCl and Taq buffer with (NH₄)₂SO₄. K⁺ stabilizes primer annealing whereas NH₄⁺ has a destabilizing effect especially on weak hydrogen bonds between mismatched primer-template base pairs. Therefore for standard PCR with Taq DNA Polymerase and 0.2 mM dNTPs the recommended MgCl₂ concentrations are in general lower 1.5±0.25 mM when using Taq buffer with KCl compared to 2.0±0.5 mM when using Taq buffer with (NH₄)₂SO₄. Due to antagonistic effects of NH₄⁺ and Mg²⁺, Taq buffer with (NH₄)₂SO₄ offers higher primer specificity in a broad range of magnesium concentrations at variety of annealing temperatures.
• For standard PCR with Pfu DNA Polymerase, 2 mM MgSO₄ is recommended.
  Volumes of 25 mM MgCl₂ or 25 mM MgSO₄ solutions required to reach a specific concentration of magnesium ions in the 50 µl reaction volume:

1. Gently vortex and briefly centrifuge all solutions after thawing.
2. Place a thin-walled PCR tube on ice and add the following components for each 50 µl reaction:
   

   10X Taq buffer	5 µl
   dNTP Mix, 2 mM each	5 µl (0.2 mM of each)
   Forward primer	0.1-1 µM
   Reverse primer	0.1-1 µM
   25 mM MgCl2*	1-4 mM
   Template DNA	10 pg – 1 µg
   Taq DNA Polymerase	1.25 u
   Water, nuclease-free	to 50 µl
   Total volume	50 µl

   * Optional for PCR with DreamTaq™ DNA Polymerase, because the provided 10X DreamTaq™ buffer contains 20 mM MgCl₂.
3. Gently vortex the samples and spin down to collect drops.

Note

• When using thermal cyclers without a heated lid, overlay the reaction mixture with 25 µl of mineral oil.
• Reaction volumes can be scaled up or down as long as the final concentrations of the reaction components remain the same.

1. Gently vortex and briefly centrifuge all solutions after thawing.
2. Place a thin-walled PCR tube on ice and add the following components for each 50 µl reaction:
   

   10X TrueStart™ Hot Start Taq buffer	5 µl
   dNTP Mix, 2 mM each	5 µl (0.2 mM of each)
   25 mM MgCl2	1.2 µl
   Forward primer	0.1-1 µM
   Reverse primer	0.1-1 µM
   TrueStart™ Hot Start Taq DNA Polymerase	0.25-2 u
   Water, nuclease-free	to 50 µl
   Total volume	50 µl

3. Gently vortex the samples and spin down to collect drops.

Note

• When using thermal cyclers without a heated lid, overlay the reaction mixture with 25 µl mineral oil.
• Reaction volumes can be scaled up or down as long as the final concentrations of the reaction components remain the same.

1. Gently vortex and briefly centrifuge all solutions after thawing.
2. Place a thin-walled PCR tube on ice and add the following components for each 50 µl reaction:
   

   10X Maxima® Hot Start Taq buffer	5 µl
   dNTP Mix, 2 mM each	5 µl (0.2 mM of each)
   Forward primer	0.1-1 µM
   Reverse primer	0.1-1 µM
   25 mM MgCl2	1-4 mM
   Template DNA	10 pg – 1 µg
   Maxima® Hot Start Taq DNA Polymerase	1.25-2 u
   Water, nuclease-free	to 50 µl
   Total volume	50 µl

3. Gently vortex the samples and spin down to collect drops.

Note

• When using thermal cyclers without a heated lid, overlay the reaction mixture with 25 µl mineral oil.
• Reaction volumes can be scaled up or down as long as the final concentrations of the reaction components remain the same.

1. Gently vortex and briefly centrifuge all solutions after thawing.
2. Place a thin-walled PCR tube on ice and add the following components for each 50 µl reaction:
   

   10X Pfu buffer with 20 mM MgSO4	5 µl
   dNTP Mix, 2 mM each	5 µl (0.2 mM of each)
   Forward primer	0.1-1 µM
   Reverse primer	0.1-1 µM
   Template DNA	50 pg – 1 µg
   Pfu DNA Polymerase	1.25 u
   Water, nuclease-free	to 50 µl
   Total volume	50 µl

3. Gently vortex the samples and spin down to collect drops.

Note

• When using thermal cyclers without a heated lid, overlay the reaction mixture with 25 µl mineral oil.
• Reaction volumes can be scaled up or down as long as the final concentrations of the reaction components remain the same.

1. Gently vortex and briefly centrifuge all solutions after thawing.
2. Place a thin-walled PCR tube on ice and add the following components for each 50 µl reaction:
   

   Components	PCR <30 kb	PCR >30 kb and GC-rich PCR
   10X long PCR buffer with 15 mM MgCl2	5 µl	5 µl
   dNTP Mix, 2 mM each	5 µl (0.2 mM of each)	5 µl (0.2 mM of each)
   Forward primer	0.3-1 µM	0.3-1 µM
   Reverse primer	0.3-1 µM	0.3-1 µM
   Template DNA	10 pg – 1 µg	1 ng – 1 µg
   DMSO	–	2 µl (4%)
   Long PCR Enzyme Mix	1.25-2.5 u	2.5 u
   Water, nuclease-free	to 50 µl	to 50 µl

3. Gently vortex the samples and spin down to collect drops.

Note

• DEPC-treat all tubes and pipette tips to be used in the cDNA synthesis or use certified nuclease-free labware.
• Use pipettes dedicated for RNA work.
• Wear gloves when handling RNA and all reagents, as skin is an common source of RNases. Change gloves frequently.
• Use certified reagents, including high quality water (e.g., nuclease-free or DEPC-treated Water).
• Use an RNase inhibitor, such as RiboLock™ RNase Inhibitor, to protect template RNA.
• Always assess the integrity of RNA prior to cDNA synthesis. For example, if sharp bands of both the human 18S rRNA (runs at approx. 1.9 kb) and the 28S rRNA (runs at approx. 5 kb) are formed during denaturing agarose gel electrophoresis of total RNA, the mRNA in the sample is considered to be intact.

1. Add to an RNase-free tube:
   

   RNA	1 µg
   10X reaction buffer with MgCl2	1 µl
   DNase I, RNase-free	1 µl (1 u)
   DEPC-treated Water	to 10 µl
   Total volume	10 µl

2. Incubate 30 min at 37°C.
3. Add 1 µl of 50 mM EDTA and incubate 10 min at 65°C. RNA hydrolyzes if heated in the absence of a chelating agent (1).
4. Use the prepared RNA as a template for reverse transcriptase.

Note

• Do not use more than 1 u of DNase I, RNase-free per µg of RNA.
• Reaction mixture can be scaled up for larger amounts of RNA. The recommended final concentration of RNA is 0.1-0.2 µg/µl.
• RiboLock™ RNase Inhibitor (#EO0381), typically at 1 u/µl, can also be included in the reaction mixture to inactivate type A RNases potentially present in the initial RNA solution

1. Add into sterile, nuclease-free tube on ice in the indicated order:

   Template RNA	total RNA or poly(A) RNA or specific RNA	10 ng – 5 µg 1-500 ng 0.01 pg – 0.5 µg
   Primer	Oligo(dT)18 or Random hexamer or Gene-specific	0.5 µg (100 pmol) 0.2 µg (100 pmol) 15-20 pmol
   DEPC-treated Water		to 12.5 µl
   Total volume	12.5 µl

2. Optional: If RNA template is GC rich or is known to contain secondary structures, mix gently, centrifuge briefly and incubate at 65°C for 5 min, chill on ice, briefly centrifuge and place on ice.
3. Add the following components in the indicated order:

   5X reaction buffer	4 µl
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   dNTP Mix, 10 mM each	2 µl (1 mM final concentration)
   RevertAid™ H Minus Reverse Transcriptase	1 µl (200 u)
   Total volume	20 µl

4. Mix gently and centrifuge briefly.
5. If oligo(dT)₁₈ primer or gene-specific primer is used, incubate 60 min at 42°C. If random hexamer primers are used, incubate 10 min at 25°C followed by 60 min at 42°C. For transcription of GC rich RNA reaction temperature can be increased to 45°C.
6. Terminate the reaction by heating at 70°C for 10 min.

Note

• The reverse transcription reaction product can be directly used in PCR or stored at -20°C.
• Use 2 µl of the reaction mix to perform PCR in 50 µl of reaction volume.

1. Prepare the following reaction mixture:
   

   Linear vector DNA	20-100 ng
   Insert DNA	1:1 to 5:1 molar ratio over vector
   10X T4 DNA Ligase buffer	2 µl
   T4 DNA Ligase	1 u
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Incubate 10 min at 22°C.
3. Use up to 5 µl of the mixture for transformation of 50 µl of chemically competent cells or use 1-2 µl per 50 µl electrocompetent cells.

Note

• The electrotransformation efficiency may be improved by:
  – heat inactivation of T4 DNA Ligase at 65°C for 10 min or at 70°C for 5 min,
  – purification of DNA, using GeneJET™ PCR Purification Kit (#K0701), or by chloroform extraction.
• The overall number of transformants may be increased by extending the reaction time to 1 hour.
• If more than 2 u of T4 DNA Ligase is used in 20 µl reaction mixture, it is necessary to purify DNA (by spin column or chloroform extraction) before electrotransformation.

1. Prepare the following reaction mixture:
   

   Linear vector DNA	20-100 ng
   Insert DNA	1:1 to 5:1 molar ratio over vector
   10X T4 DNA Ligase buffer	2 µl
   50% PEG 4000 solution	2 µl
   T4 DNA Ligase	5 u
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Incubate 1 hour at 22°C.
3. Use up to 5 µl of the mixture for transformation of 50 µl of chemically competent cells. Purify DNA for electrotransformation, using the GeneJET™ PCR Purification Kit (#K0701), o by chloroform extraction. Use 1-2 µl of DNA solution per 50 µl of electrocompetent cells.

1. Prepare the following reaction mixture:
   

   Linear DNA	10-50 ng
   10X T4 DNA Ligase buffer	5 µl
   T4 DNA Ligase	5 u
   Water, nuclease-free	to 50 µl
   Total volume	50 µl

2. Mix thoroughly, spin briefly and incubate:
   – sticky-ends for 10 min at 20°C,
   – blunt-ends for 1 hour at 22°C.
3. Use up to 5 µl of the mixture for transformation of 50 µl chemically competent cells and 1-2 µl per 50 µl of electrocompetent cells.

Note

• The electrotransformation efficiency may be improved by:
  – heat inactivation of T4 DNA Ligase at 65°C for 10 min or at 70°C for 5 min,
  – purification of DNA, using GeneJET™ PCR Purification Kit (#K0701), or by chloroform extraction.
• The overall number of transformants may be increased by extending the reaction time to 1 hour.

IMPORTANT NOTES

• Polyethylene glycol (PEG) greatly increases the ligation efficiency of blunt-end DNA ligation. The recommended concentration of PEG 4000 in the ligation reaction mixture is 5% (w/v).
• Do not exceed the recommended amount of T4 DNA Ligase in the rection mixture.
• Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X Loading Dye & SDS Solution (#R1151) at 70°C for 5 min and chill on ice prior to loading.
• For efficient transformation, the volume of the ligation reaction mixture should not exceed 10% of the competent cell volume.

1. Prepare the following reaction mixture:
   

   Linear DNA	100-500 ng
   Phosphorylated linkers	1-2 µg
   10X T4 DNA Ligase buffer	2 µl
   50% PEG 4000 solution	2 µl
   T4 DNA Ligase	2 u
   Water, nuclease-free	to 20 µl
   Total volume	to 20 µl

2. Mix thoroughly, spin briefly and incubate for 1 hour at 22°C.
3. Heat inactivate at 65°C for 10 min or at 70°C for 5 min.

Note

1. Prepare the loading mixture:

   Ligation reaction product	10 µl
   6X DNA Loading Dye & SDS Solution	2 µl

2. Heat the sample for 10 min at 65°C or for 5 min at 70°C and load.

• Appearance of higher molecular weight bands and decreased intensity of the vector and insert bands indicate successful ligation.
• Unchanged band pattern after ligation indicates unsuccessful ligation.

1. Prepare the control ligation mixture:
   

   Lambda DNA/HindIII DNA Marker (#SM0101)	1 µl (0.5 µg)
   10X T4 DNA Ligase Buffer	2 µl
   T4 DNA Ligase	1 u
   Water, nuclease-free	to 20 µl
   Total volume	to 20 µl

2. Mix thoroughly, spin briefly to collect all drops and incubate at 22°C for 10 min.
3. Prepare the loading mixture:
   

   Ligation reaction product	10 µl
   6X DNA Loading Dye & SDS Solution	2 µl

• Appearance of higher molecular weight bands and decreased intensity of the lower molecular weight bands indicates the active ligase.
• Unchanged band pattern after ligation indicates inactivated ligase.

1. Prepare the following reaction mixture:
   

   5X reaction buffer for T4 DNA Polymerase	4 µl
   Linear DNA or PCR product	1 µl
   dNTP Mix, 2 mM each	1 µl (0.1 mM final concentration)
   T4 DNA Polymerase	0.2 µl (1 u)
   Water, nuclease-free	to 20 µl
   Total volume	to 20 µl

2. Mix thoroughly, spin briefly and incubate at 11°C for 20 min or at room temperature for 5 min.
3. Stop the reaction by heating at 75°C for 10 min.

1. Prepare the following reaction mixture:
   

   Linear DNA	10-15 µl (0.1-4 µg)
   10X reaction buffer for Klenow Fragment	2 µl
   dNTP Mix, 2 mM each	0.5 µl (0.05 mM final concentration)
   Klenow Fragment	0.1-0.2 µl (1-5 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Mix thoroughly, spin briefly and incubate at 37°C for 10 min.
3. Stop the reaction by heating at 75°C for 10 min.

1. Prepare the following reaction mixture:
   

   DNA	~1 µg
   5X reaction buffer for S1 Nuclease	6 µl
   S1 Nuclease	0.1 µl (10 u)
   Water, nuclease-free	to 30 µl
   Total volume	30 µl

2. Incubate the mixture at room temperature for 30 min.
3. Stop the reaction by adding 2 µl of 0.5 M EDTA and heating at 70°C for 10 min.

Note

1. Prepare the following reaction mixture:
   

   Linear ds DNA or Oligonucleotide	1-20 pmol of 5'-termini or 10-50 pmol
   10X reaction buffer A for T4 Polynucleotide Kinase	2 µl
   ATP, 10 mM*	2 µl
   T4 Polynucleotide Kinase	1 µl (10 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

   * Prepare 10 mM ATP solution by combining 10 µl of 100 mM ATP solution and 90 µl of Water, nuclease-free.
2. Mix thoroughly, spin briefly and incubate at 37°C for 20 min.
3. Heat at 75°C for 10 min.

Note

1. Prepare the following reaction mixture:
   

   Linear DNA (~3 kb plasmid)	1 µg (~1 pmol termini)
   10X FastAP™ reaction buffer	2 µl
   FastAP™ Thermosensitive Alkaline Phosphatase	1 µl (1 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Mix thoroughly, spin briefly and incubate at 37°C for 10 min.
3. Stop reaction by heating at 75°C for 5 min.

Note

1. Prepare the following reaction mixture:
   

   Linear DNA (~3 kb plasmid)	1 µg (~1 pmol termini)
   10X SAP reaction buffer	2 µl
   Shrimp Alkaline Phosphatase (SAP)	1 µl (1 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Mix thoroughly, spin briefly and incubate at 37°C for 30 min (5'-overhangs or blunt ends) or 60 min (3'-overhangs).
3. Stop reaction by heating at 65°C for 15 min.

Note

1. Prepare the following reaction mixture containing:
   

   Plasmid DNA	1 µg
   10X FastDigest® buffer	2 µl
   FastDigest® Restriction Enzyme	1 µl
   FastAP™ Thermosensitive Alkaline Phosphatase	1 µl
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Mix thoroughly, spin briefly and incubate at 37°C for 10 min.
3. Stop reactions by heating at 65°C for 15 min or at 80°C for 20 min (if restriction enzyme is not inactivated at 65°C).

Note

Note

• The reaction can be stopped by addition of a final concentration of 50 mM EDTA (#R1021) or by addition of a final concentration of 10 mM sodium orthovanadate (Na₃VO₄).
• The optimal incubation time and the enzyme concentration must be determined experimentally for each substrate.

All Fermentas reverse transcriptases (RT) are suitable for the synthesis of full-length first strand cDNA, but they differ in reaction temperatures, amounts of RNA transcribed, sensitivity and RNase H activity. The table below indicates reaction conditions recommended for each RT. Enzyme units and RNA amounts are provided for 20 µl of RT reaction volume:

1. Add into sterile, nuclease-free tube on ice in the order given:
   

   poly(A)+ mRNA or	1 µg
   Total RNA or	5 µg
   Specific RNA transcript	0.5-1 µg
   Oligo(dT)18 primer (0.5 µg/µl) or	0.5 µg
   Random hexamer primer (0.2 µg/µl) or	0.2 µg
   Gene-specific primer	100 pmol
   DEPC-treated Water	to 11.5 µl
   Total volume	11.5 µl

2. Optional (if RNA template is GC-rich or is known to contain secondary structures). Mix gently and briefly centrifuge, incubate at 65°C for 5 min, chill on ice and briefly centrifuge, then place the tube on ice.
3. Add in the order given:

   5X reaction buffer for reverse transcriptase	4 µl
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   dNTP Mix, 10 mM each	2 µl (1 mM final concentration)
   RevertAid™ H Minus Reverse Transcriptase	1 µl (200 u)
   Total volume	20 µl

4. Mix gently and briefly centrifuge.
5. If oligo(dT)₁₈ primer or gene-specific primer is used, incubate 60 min at 42°C.
   If random hexamer primer is used, incubate 10 min at 25°C followed by 60 min at 42°C. For reverse transcription of GC-rich RNA reaction temperature can be increased up to 45°C.
6. Terminate the reaction by heating at 70°C for 10 min.

1. Add the following (on ice) to 20 µl of first strand cDNA synthesis reaction mixture:
   

   10X reaction buffer for DNA Polymerase I	8 µl
   RNase H	0.2 µl (1 u)
   DNA Polymerase I	3 µl (30 u)
   Water, nuclease-free	88.8 µl
   Total volume	100 µl

2. Gently vortex and briefly centrifuge.
3. Incubate at 15°C for 2 hours. Do not let the temperature rise above 15°C.
4. Add 2.5 µl (12.5 u) of T4 DNA Polymerase and incubate at 15°C for 5 min.
5. Terminate the reaction by adding 5 µl of 0.5 M EDTA, pH 8.0.

• When using commercial competent cells, follow the recommendations from the supplier.
• Use up to 5 µl of the ligation mixture per 50 µl of chemically competent cells including competent cells produced with TransformAid™ Bacterial Transformation Kit.
• For transformation by electroporation, extract the ligation reaction mixture with chloroform or a commercial spin column. Use 1 µl of purified ligation mixture to transform 50 µl of electrocompetent cells.
• Ligation efficiency often decreases as the insert size increases. Therefore, for vector containing large inserts (>3 kb) we recommend transformation by electroporation as it yields the highest transformation efficiencies (10⁹ cfu/µg supercoiled DNA).
• For fast cloning use competent cells prepared with the TransformAid™ Bacterial Transformation Kit. The TransformAid™ Bacterial Transformation Kit constantly yields efficiencies of 10⁷ cfu/µg supercoiled DNA.

1. When a lyophilized bacterial strain is purchased, a small portion of the powder is transferred with a sterile pipette tip or inoculating loop to liquid LB medium and incubated for 2-8 hours in a shaker.
2. A drop of liquid culture is spread on a selective plate and propagated overnight at 37°C to check for presence of selective markers.
3. Select a single colony and grow overnight.
4. Add 180 µl of 87% sterile glycerol to a 2 ml screw-cap culture vial.
5. Add 820 µl of liquid E.coli culture to vial, mix well, freeze in liquid nitrogen and store at -70°C.

1. Pour sterile warm LB agar (about 25 ml) into a Petri dish.
2. Dry opened LB plates at room temperature under UV light for about 30 min.
3. Add 40 µl of the X-Gal Solution (20 mg/ml), ready-to-use.
4. Add 40 µl of 100 mM IPTG Solution, ready-to-use.
5. Spread evenly on the plate with a sterile spatula.

1. 1 µl of X-Gal Solution (20 mg/ml), ready-to-use.
2. 1 µl of 100 mM IPTG Solution, ready-to-use.
3. Mix well.
4. Pour 25 ml of prepared LB agar into each Petri dish.
5. Dry opened LB plates at room temperature under UV light for about 30 min.

1. Prepare enough PCR master mix for the number of colonies analyzed plus one extra. For each 20 µl of reaction, mix the following reagents:
   

   Component	Taq DNA Polymerase or DreamTaq™ DNA Polymerase	PCR Master Mix (2X)
   10X Taq buffer	2 µl	-
   dNTP Mix, 2mM each	2 µl	-
   25 mM MgCl2	1.2 µl	-
   M13/pUC Reverse Sequencing primer (#SO101)	0.6 µl (10 µM)	0.6 µl (10 µM)
   M13/pUC Forward Sequencing primer (#SO100)	0.6 µl (10 µM)	0.6 µl (10 µM)
   Taq DNA polymerase	0.1 µl (0.5 u)	-
   PCR Master Mix (2X)	-	10 µl
   Water, nuclease-free	to 20 µl	to 20 µl
   Total volume	20 µl	20 µl

2. Mix thoroughly, spin briefly and aliquot 20 µl of the mix into the PCR tubes on ice.
3. Pick up an individual colony with a sterile pipette tip and resuspend it in 20 µl of the PCR master mix. Make a short strike with the same tip over culture plate to save the clone for repropagation.
4. Perform PCR: 95°C, 3 min; 94°C, 30 s, 45°C*, 30 s, 72°C 1 min/kb; 30 cycles.
5. Analyze on a gel for the presence of the PCR product of the expected length.
   * Depends on primer pair used (Tm-5).

• Add 150 µl of the overnight culture to 1.5 ml of pre-warmed C-Medium. Incubate for 20 min in a 37°C shaker.
• Prepare T-Solution – mix 250 µl of T-Solution (A) and 250 µl of T-Solution (B). Keep on ice.
• Centrifuge refreshed bacterial cells for 1 min in a microcentrifuge to pellet the cells.
• Resuspend cells in 300 µl of T-Solution. Incubate 5 min on ice.
• Centrifuge for 1 min in a microcentrifuge and resuspend cells in 120 µl of T-Solution. Incubate 5 min on ice.
• Add 1 µl of supercoiled DNA (10-100 pg) or up to 5 µl of ligation mixture (10-100 ng vector DNA) into new microcentrifuge tubes. Chill 2 min on ice.
• Add 50 µl of the prepared cells to each tube containing DNA. Incubate 5 min on ice.
• Plate immediately on pre-warmed LB-Ampicillin agar plates. Incubate overnight at 37°C.

1. Place a 0.5-1 cm sample of mouse tail into a 1.5 ml microcentrifuge tube.
2. Add 500 µl of lysis buffer (50 mM KCl, 50 mM Tris-HCl (pH 8.0), 2.5 mM EDTA, 0.45% NP-40, 0.45% Tween-20).
3. Add 2.5 µl of Proteinase K, 20 mg/ml and mix briefly.
4. Incubate overnight at 55°C.
   Optional. Incubate for an additional 1 hour at 65°C.
5. Vortex and spin 10 seconds at 13,000 rpm to collect cell debris.
6. Use 1 µl from the top part of the supernatant per 50 µl of PCR mix.

1. Centrifuge for 5 min at 3000 rpm in a microcentrifuge to collect the cells.
2. Wash the pellet twice with PBS (137 mM NaCl, 27 mM KCl, 100 mM Na₂HPO₄, 2 mM K₂HPO₄, pH 7.4).
3. Resuspend the pellet in 0.5 ml of lysis buffer (10 mM Tris-HCl, pH 8.5, 5 mM EDTA, 200 mM NaCl, 0.2% SDS). Incubate at 60°C for 5 min.
4. Add 2.5 µl of Proteinase K and 5 µl of RNase A/T1 Mix, mix. Incubate at 60°C for 1 h.
5. Add 250 µl of 5 M NaCl, mix and incubate on ice for 5 min to precipitate protein.
6. Centrifuge for 15 min at 10,000 rpm in a microcentrifuge.
7. Transfer supernatant to a fresh tube. Add an equal volume of isopropanol and mix to precipitate the DNA.
   Optional. Incubate at -20°C for up to 60 min to increase the yield of DNA.
8. Centrifuge for 10 min at 10,000 rpm in a microcentrifuge.
9. Discard the supernatant and wash the pellet with 1.2 ml 70% cold ethanol.
10. Air-dry the pellet (do not use a vacuum dryer or let the pellet dry completely. Dried genomic DNA has low solubility). Dissolve the pellet in Water, nuclease-free or TE buffer.

Note

• The typical yield of DNA from 10⁶ cells is 7-15 µg.

• Use the same 1X electrophoresis buffer to prepare the gel and to run electrophoresis.
• For analysis of linear DNA fragments add ethidium bromide to the electrophoresis buffer and gel at 0.5 µg/ml concentration. Wear gloves when handling ethidium bromide.
• For reliable in-gel analysis of circular supercoiled or relaxed plasmid DNA, do not include ethidium bromide in the electrophoresis buffer or gel. The gel should be stained only after electrophoresis.
• Ethidium bromide and exposure to UV light cause DNA alterations. Therefore, avoid UV exposure and ethidium bromide if DNA is to be purified from the gel for cloning experiments.
• For preparation of the agarose gel use a flask of at least three times larger volume than that of the solution to avoid solution loss due to boiling.

1. Weigh out the required amount (depending on the gel percentage) of agarose into an Erlenmeyer flask and add the appropriate volume of either 1X TBE or 1X TAE buffer, swirl to mix.
2. Weigh the flask with the solution.
3. For high percentage (3-5%) agarose gels: add a 10-20% excess amount of distilled water.
4. Boil the mixture in a microwave oven at medium power until the agarose is completely molten, swirl several times while boiling.
   To prepare the highest quality agarose gels of any percentage, an additional 3-5 min boiling after complete melting of the agarose is recommended.
5. Weigh the flask again and, if necessary, add hot distilled water to restore the evaporated water (or continue boiling) to obtain the correct percentage agarose gel.
   Optional. Add ethidium bromide to a final concentration of 0.5 µg/ml, mix well and heat the mixture for an additional minute without boiling.
6. Cool the solution to 65-70°C. Pour carefully on a clean casting plate with an appropriate comb. If bubbles appear, push them carefully away to the sides with a tip.
7. Allow the gel to solidify for about 30 min before using. Low percentage LM agarose gels can only be solidified at 4°C.
8. Immerse the gel into the desired electrophoresis buffer and load samples.
9. After electrophoresis the gel can be stained with ethidium bromide, SYBR® Green I or by any other staining technique.
   Warning: hot agarose solution should be handled very carefully.

1. Perform electrophoresis of DNA in low melting point (LM) agarose (#R0801) gel prepared in TAE (#B49), 0.5X TBE, TBE (#B52) or TPE buffer. Stain the gel with ethidium bromide.
2. Cut out the desired DNA band from the agarose gel with a clean scalpel under UV light*. Cut out only as much agarose as it is necessary. (The bottom of the excised agarose is free of DNA and should be removed.)
3. Determine the weight of the slice. To facilitate melting, cut gel slices larger than 200 mg into smaller pieces.
4. Incubate the tube at 70°C for approx. 10 min. Ensure that the agarose is completely melted.
5. Transfer the tube to a 42°C water bath and equilibrate for 5 min.
6. Add 1 u of Agarase (#EO0461) per 100 mg (approx. 100 µl) of molten 1% low melting agarose. Increase the amount of enzyme proportionally for higher percentage agarose, gently mix and incubate at 42°C for 30 min.
7. Add ammonium acetate** to a 2.5 M final concentration, chill on ice for 5 min.
8. Centrifuge at 10,000 rpm for 10 min to pellet undigested carbohydrates. Transfer the supernatant to a clean tube.
9. Add 2.5 volumes of ethanol or 0.8 volume of isopropanol, mix gently and incubate at room temperature for 1 h. If DNA fragments are smaller than 500 bp or if the DNA concentration is lower than 0.05 µg/ml, incubate at room temperature for 2 h.
10. Centrifuge at 10,000 rpm for 15 min, remove supernatant and dry the pellet. Resuspend the pellet in TE or another appropriate buffer for subsequent manipulation.

Note

• The procedure typically recovers 90% of DNA from the gel.
• For evaluation of DNA yield use Fermentas DNA ladders/markers, which are ideal for in-gel DNA quantification.
• T4 polynucleotide kinase is inhibited by ammonium ions, therefore use 1 M Sodium Acetate, (0.3 M final concentration) if T4 polynucleotide kinase will be used in downstream applications.
• Large DNA fragments (>30 kb) require delicate handling to avoid mechanical shearing. After digestion with agarase (step 6), centrifuge at maximum speed for 10 min to pellet undigested carbohydrates. Remove oligosaccharides and agarase by dialysis or carry out subsequent manipulations with DNA in the digested agarose solution.

1. Add 1/10 volume of 3 M Sodium Acetate (or 2 M sodium chloride, or 5 M ammonium acetate) to DNA solution.
2. Add Glycogen (#R0561 for DNA or #R0551 for RNA) to a final concentration of 0.05-1 µg/µl. Use up to 1 µl of Glycogen per 20 µl of solution.
3. Add 2.5 volumes of ethanol. Mix gently but thoroughly.
4. Incubate for 5 min at room temperature.
5. Centrifuge the mixture for 10-15 min at 10,000 rpm. Discard the supernatant.
6. Rinse the pellet with cold 70% ethanol. Air-dry the pellet.
7. DNA: Dissolve the pellet in Water, nuclease-free or TE buffer.
   RNA: Dissolve pellet in DEPC-treated Water.

1. Add 2 u of DNase I, RNase-free per 1 µg of template DNA directly to a transcription reaction mixture. In some cases, the amount of enzyme should be determined empirically.
2. Incubate at 37°C for 15 minutes.
3. Inactivate DNase I by phenol/chloroform extraction.

1. Add to an RNase-free tube:
   

   RNA	1 µg
   10X reaction buffer with MgCl2	1 µl
   DNase I, RNase-free	1 µl (1 u)
   DEPC-treated Water	to 10 µl
   Total volume	10 µl

2. Incubate 30 min at 37°C.
3. Add 1 µl of 50 mM EDTA and incubate 10 min at 65°C. RNA hydrolyzes if heated in the absence of a chelating agent (1).
4. Use the prepared RNA as a template for reverse transcriptase.

Note

• Do not use more than 1 u of DNase I, RNase-free per µg of RNA.
• Reaction mixture can be scaled up for larger amounts of RNA. The recommended final concentration of RNA is 0.1-0.2 µg/µl.
• RiboLock™ RNase Inhibitor (#EO0381), typically at 1 u/µl, can also be included in the reaction mixture to inactivate type A RNases potentially present in the initial RNA solution

1. Prepare the following reaction mixture:
   

   PCR mixture (directly after completion of PCR)	5 µl
   Exonuclease I (Exo I)	0.5 µl (10 u)
   FastAP™ Thermosensitive Alkaline Phosphatase or Shrimp Alkaline Phosphatase (SAP)	1 µl (1 u)

2. Mix well and incubate at 37°C for 15 min.
3. Stop the reaction by heating the mixture at 85°C for 15 min.

Note

• Up to 5 µl of purified PCR products can be used directly for DNA sequencing without further purification.
• For reliable sequencing results there should not be non-specific PCR products.
• The protocol may be applied for clean-up of PCR products, generated by any thermophilic DNA polymerase or polymerase mix.
• The procedure is not recommended for downstream cloning applications.

1. Mix your sample with 1 volume of Tris-saturated phenol and 1 volume of chloroform. Centrifuge at 10,000 rpm for 5 min at room temperature.
2. Transfer the upper aqueous phase to a fresh tube. Add an equal volume of chloroform and mix. Centrifuge at 10,000 rpm for 5 min at room temperature. Repeat.
3. Transfer the upper aqueous phase to a fresh tube. Add 1/10 the volume of 3 M Sodium Acetate Solution or 2 M sodium chloride.
4. Add 2.5 volumes of ethanol or an equal volume of isopropanol to precipitate DNA.
5. Incubate the mixture for 30 min at -20°C.
6. Centrifuge for 10 min at 10,000 rpm. Then discard the supernatant and rinse the pellet with 70% cold ethanol.
7. Air-dry the pellet. Dissolve in Water, nuclease-free or TE buffer.

Note

1. Add 1/10th volume of 3 M Sodium Acetate Solution to the DNA.
2. Mix thoroughly.
3. Extract with an equal volume of a 1:1 phenol/chloroform mixture, and then twice with an equal volume of chloroform. Collect the aqueous phase and transfer to a new tube.
4. Precipitate the DNA by adding 2 volumes of ethanol. Incubate at -20°C for at least 30 min and collect the pellet by centrifugation.
5. Remove the supernatant and rinse the pellet with 500 µl of cold 70% ethanol.
6. Resuspend the DNA in 20 µl of DEPC-treated water.

• Maintain a separate area, dedicated pipettors and reagents when working with RNA.
• Wear gloves when handling RNA and reagents to avoid contact with skin, which is a source of RNases. Change gloves frequently.
• Use sterile RNase free plastic tubes.
• Treat water and all solutions used for RNA purification and handling with DEPC. Add DEPC to 0.1% (v/v) final concentration; incubate overnight at room temperature and autoclave.
• High quality reagents must be used for buffer solutions. Buffers containing Tris should be prepared by dissolving Tris base in DEPC-treated water. Solutions containing DTT or nucleotides should be prepared by dissolving in DEPC-treated water and passing the solution through a 0.2 µm filter to sterilize.
• Keep all kit components sealed when not in use and all tubes tightly closed during the transcription reaction.

• Thaw frozen reagents, mix and centrifuge briefly.
• Keep enzymes and nucleotides on ice.
• Keep the Reaction Buffer at room temperature.

1. Prepare the following reaction mixture at room temperature:

   5X Transcription buffer	10 µl
   ATP/GTP/CTP/UTP Mix, 10 mM each	10 µl (2 mM final concentration)
   Linearized template DNA	1 µg
   RiboLock™ RNase Inhibitor	1.25 µl (50 u)
   T7/T3/SP6 RNA Polymerase	1.5 µl (30 u)
   DEPC-treated Water	to 50 µl
   Total volume	50 µl

2. Incubate at 37°C for 2 hours.
3. Optional: To remove template DNA add 2 µl (2 u) of DNase I, RNase-free, mix and incubate at 37°C for 15 min.
4. Stop the reaction by addition of 2 µl 0.5 M EDTA, pH 8.0 and incubate at 65°C for 10 min.

Note

1. Linearize template DNA with a restriction enzyme. Extract DNA with phenol/chloroform, then with chloroform/isoamyl alcohol, and precipitate with ethanol. Dissolve DNA in DEPC-treated Water.
2. Combine the following reaction components at room temperature in the order given:
   

   5X Transcription buffer	4 µl
   3 NTP Mix, 10 mM each* (without labeled NTP)	1 µl (0.5 mM final concentration)
   100 µM CTP	2.4 µl (12 µM final concentration)
   [alpha-32P]-CTP, ~30 TBq/mmol (800 Ci/mmol)	1.85 MBq (50 µCi)
   Linear template DNA	0.2-1.0 µg
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   T7/T3/SP6 RNA Polymerase	1 µl (20 u)
   DEPC-treated Water	to 20 µl
   Total volume	20 µl

3. Incubate at 37°C for 2 hours.
4. Stop the reaction by cooling at -20°C.
5. Determine the percentage of the label incorporated into RNA.

Note

• Expect specific radioactivity of 3-5 x10⁸ dpm/µg.
• RNA can be radiolabeled with [³²P], [³⁵S] or [³H]-ribonucleotides. Recommended amounts of radiolabeled nucleotides in 20 µl of reaction mixture are as follows: 1.85 MBq (50 µCi) for 5'-[alpha-³²P]-CTP, approx. 30 TBq/mmol (800 Ci/mmol); 11.1 MBq (300 µCi) for 5'-[alpha-³⁵S]-UTP, more than 37 TBq/mmol (1000 Ci/mmol); 0.925 MBq (25 µCi) for 5,6-[³H]-UTP, 1.1-2.2 TBq/mmol (30-60 Ci/mmol).
• The yield of the full-length transcripts is reduced when the concentration of labeled NTP is below 12 µM.

• Template DNA: may interfere with downstream applications of the RNA transcript. Template DNA should be removed by DNase I digestion directly after the transcription reaction. Add 2 u of DNase I, RNase-free, mix and incubate at 37°C for 15 min, then add 2 µl of 0.5 M EDTA, pH 8.0 and incubate at 65°C for 10 min to stop the reaction.
• Proteins and nucleotides: phenol/chloroform extraction and ethanol precipitation of RNA transcripts is recommended.

1. To 50 µl reaction mixture, add 85 µl of DEPC-treated water and 15 µl of 3 M Sodium Acetate. Mix thoroughly.
2. Extract with an equal volume of 1:1 phenol/chloroform mixture, and then twice with an equal volume of chloroform. Collect the aqueous phase and transfer to a new tube.
3. Precipitate the RNA by adding 2 volumes of ethanol. Incubate at -20°C for at least 30 min and collect the pellet by centrifugation.
4. Remove the supernatant and rinse the pellet with 500 µl of cold 70% ethanol.
5. Resuspend the RNA in 20 µl of DEPC-treated water.
6. Store the RNA at -20°C or -70°C.

Note

• Use only fresh electrophoresis buffers and freshly poured gels.
• Use clean electrophoresis chambers. For RNA gel analysis, avoid electrophoresis tanks used for DNA miniprep analysis since DNA minipreps often contain RNase A or T1.
• Use the RiboRuler™ High Range or RiboRuler™ Low Range RNA Ladder for sizing and approximate quantification of the transcript.
• Use the same loading dye for samples and RNA ladders. 2X RNA Loading Dye is available separately and is provided with all Fermentas RiboRuler™ RNA Ladders. The loading dye contains ethidium bromide for RNA visualization on denaturing formaldehyde gels.
• For native gels, add 0.5 µg/ml of ethidium bromide to the agarose gel and to the running buffer.

1. Dilute the RNA transcript with DEPC-treated water to a final concentration of 0.1-0.5 µg/µl.
2. Mix 2-4 µl (0.5-1 µg RNA) of diluted sample with an equal volume of 2X RNA Loading Dye. If using a non ready-to-use version of the RNA ladder, mix it with the loading dye solution as well. Use 0.25 µl of conventional ladder per 1 mm of the gel lane width.
3. Heat samples and ladder for 10 min at 70°C.
4. Chill samples and ladder on ice for 3 min and spin briefly prior to loading.
5. Load 1 µl of sample per 1 mm of gel lane width.
6. Run the RNA ladder in parallel with your samples. Use 0.5 µl of the ready-to-use ladder per 1 mm of gel lane width.
7. Run the gel at 5 V/cm.

• All types of DNA ends can be successfully labeled with T4 Polynucleotide Kinase. However, the labeling efficiency is greatest for the 5'-protruding DNA ends, lower for blunt ends, and is the lowest for 5'-recessed DNA ends.
• This protocol is recommended for radiolabeling of DNA markers and ladders. (Ready-to-use versions with the loading dye pre-added are not suitable for labeling).

1. Prepare the following reaction mixture:

   digested DNA	1-20 pmol of 5'-termini
   10X reaction buffer B for T4 Polynucleotide Kinase	2 µl
   [gamma-32P or gamma-33P]-ATP	40 pmol
   24% (w/v) PEG 6000 solution	4 µl
   Water, nuclease-free	to 19 µl
   T4 Polynucleotide Kinase	1 µl (10 u)
   Total volume	20 µl

2. Incubate at 37°C for 30 min.
3. Add 1 µl 0.5 M EDTA, pH 8.0 and heat at 75°C for 10 min.
4. Separate labeled DNA from unincorporated label by gel filtration on Sephadex G-50.

Note

• If an ethanol solution of [gamma-³²P or gamma-³³P]-ATP is used, dry the required amount of ATP under vacuum and dissolve in Water, nuclease-free (#R0581).
• The ATP concentration should be at least 2 µM in the exchange reaction (1, 2).
• For estimation of pmol of DNA ends, see REviewer™.

1. Prepare the following reaction mixture:
   

   Dephospharylated DNA or Oligonucleotide	1-20 pmol of 5'-termini 10-50 pmol
   10X reaction buffer A for T4 Polynucleotide Kinase	2 µl
   [gamma-32P or gamma-33P]-ATP	20 pmol
   Water, nuclease-free	to 19 µl
   T4 Polynucleotide Kinase	1 µl (10 u)
   Total volume	20 µl

2. Incubate at 37°C for 30 min.
3. Add 1 µl 0.5 M EDTA, pH 8.0 and heat at 75°C for 10 min.
4. Separate labeled DNA from unincorporated label by gel filtration on Sephadex G-50.

Note

• If an ethanol solution of [gamma-³²P or gamma-³³P]-ATP is used, dry the required amount of ATP under vacuum and dissolve in Water, nuclease-free.
• The ATP concentration should be at least 1 µM in the forward reaction (1, 2).
• For estimation of pmol of DNA ends, see REviewer™.

1. Prepare the following reaction mixture:

   RiboRuler™ Low Range RNA Ladder or RiboRuler™ High Range RNA Ladder	8 µl
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   10X reaction buffer for alkaline phosphatase	2 µl
   FastAP™ Thermosensitive Alkaline Phosphatase or Shrimp Alkaline Phosphatase	2 µl (2 u)
   DEPC-treated Water	to 20 µl
   Total volume	20 µl

2. Incubate at 37°C for 30 minutes.
3. Remove proteins from the mixture with a 20 µl aliquot of Tris-saturated (pH 8.0) phenol and chloroform mixture. Save the upper aqueous phase and extract it twice with 20 µl aliquot of chloroform.
4. Precipitate RNA by adding 1 µl of 3 M Sodium Acetate Solution, 55 µl of 96% ethanol and keep 15-30 min at -20°C. Centrifuge the mixture for 20 min at 10,000-15,000 rpm at 4°C.
5. Rinse the pellet with 20 µl cold 75% ethanol. Centrifuge 10 min at 10,000-15,000 rpm, 4°C.
6. Discard the supernatant and dissolve the air-dried pellet in 10 µl of DEPC-treated Water.

1. Prepare the following reaction mixture:

   Dephosphorylated RiboRuler™ Low Range RNA Ladder or Dephosphorylated RiboRuler™ High Range RNA Ladder	1 µl 2.5 µl
   [gamma-32P]-ATP (5000 Ci/mmol,10 µCi/µl)*	5 µl (10 pmol)
   RiboLock™ RNase Inhibitor	0.25 µl (10 u)
   10X buffer A for forward reaction (supplied with T4 polynucleotide kinase)	1 µl
   T4 Polynucleotide Kinase	1 µl (10 u)
   DEPC-treated Water	to 10 µl
   Total volume	10 µl

   * If [gamma-³²P]-ATP with a high specific activity (higher than 5000 Ci/mmol) is used, the label can be diluted with ATP. Total ATP concentration should be at least 1 µM.
2. Incubate at 37°C for 30 minutes.
3. Stop the reaction by adding 1 µl of 0.5 M EDTA, pH 8.0 and extract the mixture with an equal volume of chloroform.
4. Determine the efficiency of label incorporation.
5. Load the ladder on the gel.

1. Prepare the following reaction mixture:

   Linear DNA (aqueous solution)	0.1-4 µg
   10X reaction buffer for Klenow Fragment or 10X Bsm buffer	2 µl
   [alpha-32P]-dNTP, ~15-30 TBq/mmol (400-800 Ci/mmol) or [alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	0.74 MBq (20 µCi) 2.96 MBq (80 µCi)
   3 dNTP Mix, 2 mM each (without a labeled dNTP)	2.5 µl (0.25 mM final concentration)
   Klenow Fragment or Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment	0.1 µl (1 u) 0.2 µl (1 u) 0.125 µl (1 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Incubate at 37°C for 15 min.
3. Stop the reaction by heating at 75°C for 10 min.

Note

• The modified version of this protocol can be used for non-radioactive labeling of DNA markers. Substitute a part of dTTP nucleotide with a modified nucleotide (e.g. Biotin-11-dUTP or Fluorescein-12-dUTP) at a molar ratio of 1:2.
• For estimation of pmol of DNA ends, see REviewer™.

1. Prepare the following reaction mixture:

   5X reaction buffer for Terminal Deoxynucleotidyl Transferase	10 µl
   Linear DNA	10 pmol
   [alpha-32P]-ddATP, ~110 TBq/mmol (3000 Ci/mmol)	1.85 MBq (50 µCi)
   Terminal Deoxynucleotidyl Transferase	2 µl (40 u)
   Water, nuclease-free	to 50 µl
   Total volume	50 µl

2. Incubate at 37°C for 15 min.
3. Stop the reaction by heating at 70°C for 10 min or by the addition of 5 µl 0.5 M EDTA.

Note

1. Prepare the following in a single RNase-free microfuge tube:

   10X ligation buffer for T4 RNA Ligase	2 µl
   10 mM ATP	1 µl
   RNA	50-100 pmol
   [32P]-pCp	50-100 pmol (equimolar amount)
   T4 RNA Ligase	1 µl (10 u)
   DEPC-treated Water	to 20 µl
   Total volume	20 µl

2. Incubate at 4°C for 10-12 hours (overnight).
3. Separate labeled RNA from unincorporated label by gel filtration on Sephadex G-50.

1. Prepare the following reaction mixture:

   DNA (aqueous solution)	10 µl (100 ng)
   10X reaction buffer for Klenow Fragment, exo- or 10X Bsm buffer	5 µl
   6.0 A260units/ml (100 µM) Random Hexamer Primer	12.5 µl
   Water, nuclease-free	to 40 µl
   Total volume	40 µl

2. Incubate the mixture in a boiling water bath for 5-10 minutes and then chill on ice.
3. Add:

   3 dNTP Mix, 0.33 mM each (without a labeled dNTP)	3 µl (0.02 mM final concentration)
   [alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	1.85 MBq (50 µCi)
   Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment	1 µl (5 u) 1 µl (8 u)
   Water, nuclease-free	to 50 µl
   Total volume	50 µl

4. Incubate the reaction mixture for 10 minutes at 37°C.
5. Add 4 µl 0.25 mM dNTP mix and incubate at 37°C for 5 minutes.
6. Add 1 µl 0.5 M EDTA, pH 8.0 to stop the reaction.
7. Remove 1 µl of the reaction mixture and determine the percentage of label incorporated.
8. Purify by using Sephadex G-50 or Bio-Gel P-60.

1. Prepare the following reaction mixture:

   DNA template	10 µl (100 ng – 1 µg)
   10X reaction buffer for Klenow Fragment, exo-	5 µl
   6.0 A260units/ml (100 µM) Random Hexamer Primer	12.5 µl
   Water, nuclease-free	to 39 µl
   Total volume	39 µl

2. Incubate the mixture in a boiling water bath for 5-10 minutes and then chill on ice.
3. Add:

   3 dNTP Mix, 1 mM each (without the dTTP)	5 µl (0.1 mM final concentration)
   dTTP	3.25 µl (0.065 mM final conc.)
   Biotin-11-dUTP*, 1 mM	1.75 µl
   Klenow Fragment, exo-	1 µl (5 u)
   Total volume	50 µl

   * Fluorescein-12-dUTP, DIG-dUTP or Aminoallyl-dUTP can also be used with the same protocol.
4. Incubate the reaction mixture at 37°C for 1 hour.
5. Add 1 µl 0.5 M EDTA, pH 8.0 to stop the reaction.
6. Remove 1 µl of the reaction mixture and determine the percentage of label incorporated.
7. Optionally, purify by using Sephadex G-50 or Bio-Gel P-60.

I. Radioactive DNA labeling by nick-translation

1. Mix the following components:

   10X reaction buffer for DNA Polymerase I	2.5 µl
   Mixture of 3 dNTPs, 1 mM* (without the labeled dNTP)	1.25 µl
   [alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	1.85-3.7 MBq (50-100 µCi)
   DNase I, RNase-free freshly diluted to 0.002 u/µl**	1 µl
   DNA Polymerase I	0.5-1.5 µl (5-15 u)
   Template DNA	0.25 µg
   Water, nuclease-free	to 25 µl
   Total volume	25 µl

2. Immediately incubate at 15°C for 15-60 minutes.
3. Terminate the reaction by adding 1µl of 0.5 M EDTA, pH 8.0.
4. Take an aliquot (1 µl) to determin the efficiency of the label incorporation. A specific activity of DNA at least 10⁸ cpm/µg DNA is expected.
5. If needed, the labeled DNA may be separated from the unincorporated radioactive precursors on Sephadex G-50 or Bio-Gel P-60 column or using spin column (e.g. GeneJET™ PCR Purification Kit (#K0701).

Note

• The reaction volumes can be scaled up or down providing that the final concentrations of the components (DNA, dNTPs, labeled dNTP) are as indicated in the protocol.
• Radioactive DNA probes with higher specific activities can be prepared using two radioactively labeled dNTPs simultaneously. In this case, the composition of the unlabeled dNTP mix should be adjusted accordingly.

II. Non-radioactive DNA labeling by nick-translation
The protocol above can be used for non-radioactive labeling by nick-translation using biotin-11-dUTP, fluorescein-12-dUTP, DIG-dUTP or aminoallyl-dUTP:

• normal dTTP is subsituted for labeled-dUTP at a molar ratio of 1:3-1:5,
• reaction time is prolonged to 1-2 hours.

1. Linearize template DNA with a restriction enzyme. Extract DNA with phenol/chloroform, then with chloroform/isoamyl alcohol, and precipitate with ethanol. Dissolve DNA in DEPC-treated Water.
2. Combine the following reaction components at room temperature in the order given:

   DEPC-treated Water	to 20 µl
   5X transcription buffer for RNA Polymerase	4 µl
   3 NTP Mix, 10 mM each* (without labeled NTP)	1 µl (0.5 mM final concentration)
   100 µM CTP	2.4 µl (12 µM final concentration)
   [alpha-32P]-CTP, ~30 TBq/mmol (800 Ci/mmol)	1.85 MBq (50 µCi)
   Linearized template DNA	0.2-1.0 µg
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   T7 RNA Polymerase or SP6 RNA Polymerase or T3 RNA Polymerase	1 µl (20 u)
   Total volume	20 µl

3. Incubate at 37°C for 2 hours.
4. Stop the reaction by cooling at -20°C.
5. Determine the percentage of the label incorporated into RNA.

Note

• Expect specific radioactivity of 3-5 x10⁸ dpm/µg.
• RNA can be radiolabeled with [³²P], [³⁵S] or [³H]-ribonucleotides. Recommended amounts of radiolabeled nucleotides in 20 µl of reaction mixture are as follows:
  1.85 MBq (50 µCi) for 5'-[alpha-³²P]-CTP, approx. 30 TBq/mmol (800 Ci/mmol);
  11.1 MBq (300 µCi) for 5'-[alpha-³⁵S]-UTP, more than 37 TBq/mmol (1000 Ci/mmol);
  0.925 MBq (25 µCi) for 5,6-[³H]-UTP, 1.1-2.2 TBq/mmol (30-60 Ci/mmol).
• The yield of the full-length transcripts is reduced when the concentration of labeled NTP is below 12 µM.

I. Synthesis of cDNA probes with high specific radioactivity

• This protocol is provided for first strand cDNA synthesis using RevertAid™ H Minus Reverse Transcriptase. For specific reaction conditions using other enzymes, see Table below. Enzyme units and RNA amounts are provided for 20 µl of RT reaction volume:
  

  Reverse transcriptase	Reaction tmp.	Active up to	Reading length	RHase H activity	Inactivation	Units	Total RNA	poly(A) RNA
  Maxima® RT	50-55°C	60°C	20kb	+	85°C, 5min	200	1pg-5µg	0.1pg-500ng
  RevertAid™ Premium RT	50-55°C	60°C	20kb	–	85°C, 5min	200	1pg-5µg	0.1pg-500ng
  RevertAid™ H Minus RT	42-45°C	55°C	13 kb	–	70°C, 10min	200	0.1ng-5µg	10pg-500ng
  RevertAid™ RT	42°C	50°C	13kb	+	70°C, 10min	200	0.1ng-5µg	10pg-500ng
  M-MuLV RT	37°C	37°C	9kb	+	70°C, 10min	40	100ng-5µg	10-500ng
  AMV RT	45-60°C	60°C	13kb	++	85°C, 5min	10	10ng-5µg	1-100ng

• Mix and briefly centrifuge all components after thawing, keep on ce.

1. Add into sterile, nuclease-free tube on ice in the order given:

   Template RNA	Total RNA or	up to 5 µg
   	Poly(A) RNA or	up to 500 ng
   	Specific RNA or	up to 500 ng
   Primers	Oligo(dT)18 or	0.5 µg (100 pmol)
   	Random Hexamer	0.2 µg (100 pmol)
   Gene-specific		15-20 pmol
   DEPC-treated Water		to 8.5 µl
   Total volume		8.5 µl

2. Mix gently and centrifuge to collect all drops.
   Optional. Incubate at 65°C for 5 min, chill on ice and briefly centrifuge to collect drops. Perform this step if RNA template is GC-rich or is known to contain secondary structures.
3. Place the tube with primer/template mix on ice and add the following components in the indicated order:

   5X reaction buffer	4 µl
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   dGTP, dCTP, dTTP mix, 10 mM each	1 µl
   0.1 mM dATP	4 µl
   [alpha-32P]-dATP, 3000 Ci/mmol	1 µl
   RevertAid™ H Minus Reverse Transcriptase	1 µl (200 u)
   Total volume	20 µl

4. Mix gently and centrifuge to collect all drops.
5. If Oligo(dT)₁₈ primer or gene-specific primer is used, incubate 60 min at 42°C. If random hexamer primer is used, incubate 10 min at 25°C followed by 60 min at 42°C. For transcription of GC-rich RNA reaction temperature can be increased to 45°C.
6. Stop the reaction by adding 5 µl of 0.5 M EDTA, pH 8.0.
   Optional. Hydrolyze RNA by the addition of equal volume (25 µl) of 0.6 M NaOH and incubation at 70°C for 30 min.
7. Remove unincorporated dNTPs by chromatography on a Sephadex® G-50 column.
8. Expect specific radioactivity of >10⁷ dpm/µg.

Note

• To achieve higher specific activities (over 10⁸ dpm/µg), use up to 100 µCi of [alpha-³²P]-dATP in the labeling mixture. To keep the total reaction volume of 20 µl, vacuum-evaporate 10 µl of [alpha-³²P]-dATP (10 mCi/ml) to 1 µl in a separate tube.

II. Synthesis of non-radioactively labeled cDNA

• normal dTTP is subsituted with labeled-dUTP at a molar ratio of 1:3-1:4,
• reaction time is prolonged to 2-6 hours.

1. Denaturation solution: 1.5 M NaCl, 0.5 M NaOH.
2. Neutralization solution: 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2), 1 mM EDTA.
3. 20X SSC, pH 7.0 (blotting buffer): 3 M NaCl, 0.3 M sodium citrate, 1 mM EDTA.
4. 100X Denhardt\'s solution: 2% (w/v) BSA, 2% (w/v) Ficoll™, 2% (w/v) PVP (polyvinylpyrrolidone).
5. Pre-hybridization solution: 6X SSC, 5X Denhardt's solution, 50% formamide, 0.5% SDS.

1. Rinse the gel in deionized water, add Denaturation solution and shake for 30 min at room temperature. Rinse the gel in deionized water and add Neutralization solution. Shake for 15 min at room temperature. Repeat neutralization procedure.
2. Fill the glass dish with 20X SSC blotting buffer. Make a platform and cover it with a sheet of Whatman™ 3 MM filter paper, saturated with the blotting buffer (see picture below).
3. Place the gel upside down on the filter and avoid trapping air bubbles beneath it.
4. Cut a sheet of SensiBlot™ Plus Nylon Membrane to match the size of the gel and place it on the top of the gel. Avoid trapping air bubbles beneath the membrane.
5. Cut 2-3 sheets of Whatman™ 3 MM filter paper to the size, wet with blotting buffer and place on the top of the membrane.
6. Place a stack of absorbent paper towels on top of the 3 MM paper, place a glass plate on the top of the paper towels and put a 0.5 kg weight on the top.
7. Allow upward capillary transfer of DNA at room temperature for 18 hours.
8. Wash the membrane in 2X SSC buffer to remove any residual agarose, dry at room temperature and fix for 2 min under UV-light.

1. Prepare several dilutions of the labeled probe (from 1 ng/µl to 10 fg/µl) and spot 1 µl of each dilution onto a nylon membrane strip.
2. Air-dry the spotted membrane at room temperature for 30-45 minutes or at 80°C for 10 minutes.
3. Place the membrane on a UV trans-illuminator (spotted side down) and cross link the probe to the membrane for 1-5 minutes.

Note

1. Hybridization probe for the genomic DNA (test probe).
2. Hybridization probe for visualization of DNA Marker (e.g. DNA Markers for Genomic DNA analysis). 50 ng of marker is sufficient for generation of radioactively labeled probe for 3-5 hybridization reactions.

1. Prepare 30 ml of the pre-hybridization solution.
2. Denature sonicated salmon sperm DNA solution (10 mg/ml) by heating at 100°C for 5 min. Chill on ice and add to the pre-hybridization solution to a final concentration of 50-100 µg/ml.
3. Place the membrane into the hybridization container, add pre-hybridization solution with the denatured salmon sperm DNA (0.2 ml/cm² of membrane) and pre-hybridize for 2 hours at 42°C with shaking.
4. Prepare the hybridization solution:
   • mix the two prepared probes: labeled probe for the DNA marker and probe for genomic DNA. Denature by heating at 100°C for 5 min and chill immediately on ice.
5. Add the following amounts of the probe mixture to the pre-hybridization solution:
   • to 10 ng/ml (1/5 of probe mix) if specific activity is 10⁸ dpm/µg,
   • to 2 ng/ml (1/25 of probe mix) if specific activity is 10⁹ dpm/µg,
   • to 25-100 ng/ml if non-radioactively labeled probes.
6. Discard the pre-hybridization solution (from step 3) and add the prepared hybridization solution to the hybridization bag (60 µl/cm²). Incubate for at least 12 hours at 42°C.
7. Carry out the following washes of the membrane:
   • twice in 2X SSC + 0.1% SDS for 10 min at room temperature,
   • twice in 0.1X SSC + 0.1% SDS for 10 min at 65°C (for high stringency).
8. Dry the membrane using sheets of Whatman™ 3 MM paper.

• Low percentage gels are recommended for analysis of large proteins and high percentage gels for analysis of small proteins.
• Linear gradient gels allow for high resolution of a broad range of both small and large proteins.
  Gel Recommendations.

  Protein MW range, kDa	Recommended gel, %
  ~5-50	18
  ~5-60	16
  ~10-80	14
  ~20-150	12
  ~30-200	10
  ~40-250	8
  ~60-300	6
  ~100-400	4
  Protein MW range, kDa	Recommended gradient gel, %
  ~5-100	10-20
  ~5-300	4-20
  ~10-200	8-16
  ~30-300	4-12

• All Fermentas protein ladders/markers can be used on 6, 8, 10, 12, 14 % SDS polyacrylamide gels and on 4-12%, 8-16%, 4-20% and 10-20% gradient gels.
• The following general rule can be applied to protein ladders/markers as well as to protein samples:
  • in low percentage gels (4-8%), small proteins (10-15 kDa) migrate with the tracking dyes during electrophoresis and may be not visible;
  • in high percentage gels (14-18%) large proteins (150-250 kDa) may not separate.
• For more precise determination of molecular weights, unstained protein ladders/markers are recommended.
• Prestained standards are ideal for monitoring the process of electrophoresis and the protein transfer efficiency in Western blotting.
• Prestained proteins may have different mobilities in various SDS-PAGE buffer and gel systems due to coupled chromophores that affect protein mobility. Prestained standards are recommended when approximate sizing is enough.
• Each lot of prestained protein ladder/marker is calibrated against a precisely sized unstained protein ladder/marker in Tris-glycine-SDS gel and the calculated apparent molecular weights are reported in the product's Certificate of Analysis. The prestained protein may have different mobility in other electrophoresis buffer and gel systems.
• Modifications to native proteins such as phosphorylation and glycosylation may alter protein mobility. The molecular weights of modified proteins may not correspond to those of unmodified proteins of the same size.

1. Thaw the ladder either at room temperature or at 37°C for a few minutes to dissolve precipitated solids. Do not boil.
2. Mix gently, but thoroughly, to ensure that the solution is homogeneous.
3. For Unstained Protein Molecular Weight Marker only:
   • transfer the required aliquot to a clean tube with a screw cap;
   • heat at 95°C for 10 minutes;
   • cool and mix.
     Once denatured the marker can be further used just after the thawing.
4. Load the following volumes of the ladder/marker on SDS-polyacrylamide gel with a thickness of 0.75 mm:
   • 5 µl per well for mini-gels;
   • 10 µl per well for large gels.
     For Spectra™ Multicolor Broad Range Protein Ladder:
   • 10 µl per well for mini-gels;
   • 20 µl per well for large gels.

Note

• To avoid overloading the gel which will be subsequently silver stained, dilute the ladder/marker just prior to use:
  Water, nuclease-free: 36.5 µl
  5X Protein Loading Buffer*: 10 µl
  20X Reducing Agent (#R0891): 2.5 µl
  Protein ladder/marker: 1 µl
• * Alternatively 4X DualColor™ Protein Buffer Loading Pack can be used. Volumes of the buffer and water should be adjusted appropriately.
• Staining is not required to visualize prestained protein ladders/markers.
  To visualize unstained protein ladders/markers, the gel can be processed with PageBlue™ Protein Staining Solution, PageSilver™ Silver Staining Kit or other protein staining techniques. For silver staining, the volume of Unstained Protein Molecular Weight Marker used should be decreased up to 10-fold.

1. Assemble the glass plate sandwich. Prepare the resolving gel solution as described above.
2. Add APS and TEMED last, mix carefully to avoid formation of bubbles.
3. Important Note. Polymerization begins as soon as APS is added to the mixture, so all subsequent actions must be performed promptly.
4. Pour the gel solution between the glass plates with a pipette, leave about 1/4 of the space free for the stacking gel. Carefully cover the top of the resolving gel with 50% isopropanol, 0.1% SDS solution or water, and wait until the resolving gel polymerizes (~30 min). A clear line will appear between the gel surface and the solution on top when polymerization is complete.
5. Discard the water, isopropanol or SDS solution. Wash gently with double-distilled water.
6. Pour the stacking gel solution (prepared as described above, add APS and TEMED last) carefully with a pipette to avoid formation of bubbles.
   Important Note. Polymerization begins as soon as APS is added to the mixture, so all subsequent actions must be performed promptly.
7. Insert combs. Allow the gel to polymerize for at least 60 min.
8. Remove combs carefully. Put the gel into the electrophoresis tank, fill the tank (bottom and top reservoirs) with fresh 1X Tris-glycine-SDS Buffer, make sure that the gel wells are covered with the buffer.
9. Load protein ladder/marker and probes.
10. Set an appropriate voltage and current depending on how many gels you run. Increase the power when the dye front reaches the running gel. For exact values refer to the table below:

    Gel	1 minigel	2 minigels
    Stacking gel (upper)	13 mA	25 mA
    Resolving gel (lower)	25 mA	50 mA

    Values presented are for 0.75 mm gels. For thicker gels the current should be appropriately increased.
11. Stop the electrophoresis run when the dye front reaches the bottom of the gel. Disassemble the gel sandwich and proceed with gel staining or Western blot procedures.

Note

• PageBlue™ Protein Staining Solution can be reused up to 3 times without a decrease in sensitivity.
• All reagent volumes are for 8x10 or 10x10 cm minigels of 0.75-1 mm thickness. Gels should be completely immersed in solution.
• When several gels are being stained, increase the amount of staining solution accordingly.
• The first wash step is crucial to remove SDS from the gel as SDS interferes with the staining reaction.
• For staining native gels without SDS, the washing step is not required.
• Staining sensitivity can be increased if the proteins are fixed for 15 min either with 12% trichloracetic acid or with 25% isopropanol supplemented with 10% acetic acid. Fixation prevents protein diffusion from the gel and accelerates SDS removal. After fixation, gels can be stained immediately without additional washing.
• Using either the fast or conventional protocol, staining sensitivity is 5 ng of protein per band. To increase sensitivity to 0.05 ng per band the gel can be stained using the PageSilver™ Silver Staining Kit.
• For staining peptides or small proteins (more than 10 kDa) fixation of the proteins for 15 min either with 12% trichloracetic acid or with 25% isopropanol supplemented with 10% acetic acid is recommended. Fixation prevents protein diffusion from the gel and accelerates SDS removal. After fixation, gels can be stained immediately without additional washing. Overnight staining time is required for peptide detection.

Note

1. Presoak 2-4 pieces of blotting paper (cut to the size of the gel) in transfer buffer for 5 min.
2. Cut a piece of nitrocellulose membrane to the size of the gel and equilibrate it in transfer buffer. If a PVDF membrane is used, incubate it in methanol for 2 min before equilibrating it in transfer buffer. Use CAPS buffer for N-terminal sequencing, Tris-glycine-methanol protein transfer buffer is suitable for all other applications.
3. Carefully remove the stacking gel from the resolving gel. Soak the resolving gel in CAPS buffer for 5 min if this buffer is used. This step can be omitted if Tris-glycine-methanol protein transfer buffer is used.
4. Assemble the transfer sandwich with the resolving gel on the anode (+) as shown in Figure below. Use one sheet of blotting paper or two pieces of filter paper on each side of the sandwich. Make sure all air bubbles are removed since they will affect the efficiency of the electroblotting.
5. Electrotransfer proteins from the gel on the membrane for ~60 min at room temperature. Maintain the current at 0.8 mA per 1 cm² of the gel area and limit the voltage to 15 V.
6. When the transfer is complete, turn off the power and peel off the layers of the sandwich until you reach the membrane. Remove the membrane with forceps and rinse it in deionized water.

Note

1. Add PageBlue™ Protein Staining Solution to cover the PVDF membrane. Agitate gently for 2 min.
2. Wash with 30% ethanol with gentle agitation for 5 min.

Protein fixation with glutaraldehyde is required before staining the gel with Coomassie Brilliant Blue dye (PageBlue™ Protein Staining Solution, #R0571) or silver (PageSilver™ Silver Staining Kit, #K0681).

Note

Procedure

1. Add 100 ml of deionized or distilled water to the gel and wash for 1 min with gentle agitation. Discard the wash.
2. Add 50 ml of freshly prepared 5% glutaraldehyde solution (gel should be covered completely). Fix with gentle agitation for 30 min. Discard the solution.
3. Add 100 ml of deionized or distilled water to the gel and wash for 5 minutes with gentle agitation. Discard the wash. Repeat this step twice. The gel is now ready for staining.

For detailed staining protocols using Fermentas PageBlue™ Protein Staining Solution or PageSilver™ Silver Staining Kit, refer to the product manuals.

Note

• higher Tris concentration in gel buffer (0.75 M instead of 0.375 M);
• pH of 8.45 for both stacking and resolving gels;
• higher acrylamide cross-linking in resolving gel (C= 5% instead of the usual ~3%);
• ethylene glycol is included in the resolving gel.

Reagents

• Ethylene glycol
• Stock solutions of acrylamide/bisacrylamide 19:1 and 29:1
• 3 M Tris-HCl buffer containing 0.4% SDS, pH 8.45
• 40% ammonium persulfate (APS)
• TEMED
• 1X Tris-tricine-SDS Buffer (10X Buffer (#B48), diluted to 1X concentration prior to use)

Gel Preparation
The protocol is sufficient for two 0.75 mm mini gels.

Note

Procedure (for 0.75 mm minigel):

1. Pour 3.3 ml of the resolving gel solution between the glass plates with a pipette. Quickly but carefully apply 1.1 ml of the stacking gel solution. Avoid the formation of the bubbles.

   Note

   Because of the difference in densities of the stacking and resolving gel solutions, they do not mix with each other; a sharp interface is obtained immediately after applying the stacking gel solution.
2. Insert the comb. Ensure that no air bubbles are left in the gels. Allow the gels to polymerize for 1 hour at room temperature.

   Note

   For best results, keep the gel at 4°C overnight in a plastic bag with some electrophoresis running buffer (to avoid drying). Do not remove the combs.
3. Just prior to using the gel, remove the comb carefully. Place the gel into the electrophoresis tank, fill the tank (bottom and top reservoirs) with fresh 1X Tris-tricine-SDS buffer, making sure that the gel wells are covered with the buffer.
4. Load the samples.
5. Perform electrophoresis at 200 V for ~2 hours or until the dye front reaches the bottom of the gel.
6. Disassemble the gel sandwich and proceed with gel staining or Western blot procedures.

• Use the same DNA loading dye (supplied with the DNA ladder/marker) for both the sample DNA and the ladder/marker DNA.
• If possible, always load equal volumes of the sample DNA and the ladder/marker DNA. The sample can be diluted with 1X DNA loading dye.
• Avoid high salt concentrations in the DNA samples as this may cause bands to shift during electrophoresis.
• Following electrophoresis, visualize DNA by staining in 0.5 µg/ml ethidium bromide solution or SYBR® Green I.
• Choose the gel percentage according to the tables below:
  Table 1. Recommended Agarose Gels for Electrophoretic Separation of DNA Fragments.

  Agarose gel, %	Range of effective separation, bp	Approximate positions of tracking dyes, bp*
  		Bromophenol blue		Xylene cyanol FF
  		TBE buffer	TAE buffer	TBE buffer	TAE buffer
  0.5	2000-50000	750	1150	13000	16700
  0.6	1000-20000	540	850	8820	11600
  0.7	800-12000	410	660	6400	8500
  0.8	800-10000	320	530	4830	6500
  0.9	600-10000	260	440	3770	5140
  1.0	400-8000	220	370	3030	4160
  1.2	300-7000	160	275	2070	2890
  1.5	200-3000	110	190	1300	1840
  2.0	100-2000	65	120	710	1040
  3.0	25-1000	30	60	300	460
  4.0	10-500	18	40	170	260
  5.0	10-300	12	27	105	165

  Table 2. Recommended Polyacrylamide Gels for Electrophoretic Separation of DNA Fragments (1).

  Polyacrylamide gel (with BIS at 1:20), % (w/v)	Range of effective separation*	Approximate positions of tracking dyes*
  		Bromophenol blue	Xylene cyanol FF
  Denaturing gels
  4.0	100-500 b	50 b	230 b
  5.0	70-400 b	35 b	130 b
  6.0	40-300 b	26 b	105 b
  8.0	30-200 b	19 b	75 b
  10.0	20-100 b	12 b	55 b
  15.0	10-50 b	10 b	0 b
  20.0	5-30 b	8 b	28 b
  30.0	1-10 b	6 b	20 b
  Non-denaturing gels
  3.5	100-1000 bp	100 bp	460 bp
  5.0	80-500 bp	65 bp	260 bp
  8.0	60-400 bp	45 bp	160 bp
  12.0	50-200 bp	20 bp	70 bp
  15.0	25-150 bp	15 bp	60 bp
  20.0	5-100 bp	12 bp	45 bp

Note

• Only high purity agarose should be used. TopVision™ Agarose was used to prepare the gels.
• Only freshly prepared electrophoresis buffers should be used. The buffers were prepared from Fermentas 50X TAE Buffer and 10X TBE Buffer.

• Choose electrophoresis conditions according to the recommendations below:

  Size of the DNA	Voltage	Buffer
  <1 kb	5-10 V/cm	TBE
  1-5 kb	4-10 V/cm	TAE or TBE
  > 5 kb	1-3 V/cm	TAE
  Up to 10 kb, fast electrophoresis with Express DNA ladders	up to 23 V/cm	TAE

• Always use the same DNA loading dye (supplied with the DNA ladder/marker) for both the sample DNA and the ladder/marker DNA.
• Always compare the sample band with the ladder band of the closest size.
• If possible, adjust the concentration of the sample to approximately equalize it with the amount of DNA in the nearest band.
• dNTPs, oligonucleotides, genomic DNA, RNA, NTPs or buffer components can interfere with spectrophotometrical measurements and lead to inaccurate quantification of sample DNA. In these cases, it is best to rely on gel quantification data.
• For the most accurate quantification, use video-densitometry analysis.

1. Add 1 volume of 6X DNA loading dye to 5 volumes of DNA sample.
2. Mix well, spin down and load.

• probes after DNA restriction digestions, ligation or dephosphorylation reactions,
• DNA samples with high amounts of DNA binding proteins,
• DNA molecules with cohesive ends,
• or to stop an enzymatic reaction during kinetic experiments.

1. Add 1 volume of 6X DNA Loading Dye & SDS Solution to 5 volumes of DNA sample.
2. Mix well.
3. Heat at 65°C for 10 minutes.
4. Chill on ice, spin down and load. 0

Note

Note

1. Mix the DNA sample with an equal volume of 2X RNA Loading Dye.
2. Heat at 95°C for 5 min.
3. Chill the sample on ice for 3 min.
4. Keep samples on ice while loading.

Note

• Use a flask of at least three times larger volume than that of the solution to avoid boiling over.
• Use the same 1X electrophoresis buffer to prepare the gel and to run electrophoresis.
• Dilute 50X TAE Buffer or 10X TBE Buffer to a 1X concentration immediately before use.
• Use TBE buffer for analysis of DNA bands smaller than 1500 bp. For larger DNA, use TAE buffer.
• For intensified gel staining, add ethidium bromide to both the gel and the electrophoresis buffer at a final 0.5 µg/ml concentration. Alternatively, stain the gel after electrophoresis (see below).
  Wear gloves when handling ethidium bromide.
• For reliable analysis of supercoiled/relaxed plasmid ethidium bromide should not be included in the electrophoresis buffer or gel. The gel should be stained only after electrophoresis is complete.
• Ethidium bromide and exposure to UV light may cause DNA alterations. Therefore, avoid UV exposure and do not stain DNA with ethidium bromide if the purified fragments will be used for cloning experiments.

1. Weigh out the required amount of agarose (depending on the gel percentage) into an Erlenmeyer flask.
2. Add the appropriate volume of either 1X TBE or 1X TAE buffer and swirl to mix.
3. Weigh the flask with the solution.
   For high percentage gels (3-5%): add an excess amount of distilled water to increase the weight by 10-20%.
4. Boil the mixture in a microwave oven (at medium power) until the agarose melts completely; swirl the flask several times while boiling. To prepare the highest quality agarose gels of any percentage, an additional 3-5 min of boiling after completely melting the agarose is recommended. A significant amount of water evaporates during this procedure and therefore restoring of the initial weight (in step 5) is required to obtain the desired percentage gel.
5. Weigh the flask again and if necessary, add hot distilled water to restore the initial weight.
   For high percentage gels (3-5%): check (by weighing) that the excess 10-20% of water has evaporated and, if needed, continue boiling to remove any excess, or add hot distilled water to restore the initial weight.
   Optional: for intensified gel staining add ethidium bromide to a final concentration of 0.5 µg/ml. Mix well and heat for 1 min without boiling.
6. Cool the solution to 65-70°C. Pour carefully on a clean casting plate with an appropriate comb. If bubbles appear, push them carefully away to the sides with a pipette tip.
7. Solidify the gel for approximately 30 min before use. Low percentage LM agarose gels need to be solidified at 4°C.
8. Immerse the gel into the desired electrophoresis buffer. Load the samples onto the gel.
9. Run electrophoresis at 5-7 V/cm until the bromophenol blue runs approximately two-thirds of the way down the gel.
10. After electrophoresis the gel can be stained by immersing it into a 0.5 µg/ml ethidium bromide solution for 15-20 min, stained with SYBR® Green I or any other DNA staining technique.
    Warning. Hot agarose solution should be handled very carefully.

Note

• Double stranded DNA ladders are not recommended for denaturing electrophoresis as they may form an atypical pattern. However, these discrepancies are normally acceptable for analysis of cDNA or other ssDNA in alkaline gels.
• Use a flask of at least three times larger volume than that of the solution to avoid boiling over.
• Wear gloves when handling ethidium bromide.

1. Weigh out the required amount of agarose (depending on the gel percentage) into an Erlenmeyer flask.
2. Add the appropriate volume of the buffer (30 mM NaCl, 2 mM EDTA, pH 7.5) and swirl to mix.
3. Weigh the flask with the solution.
   For high percentage gels (3-5%): add an excess amount of distilled water to increase the weight by 10-20%.
4. Boil the mixture in a microwave oven (at medium power) until the agarose melts completely; swirl the flask several times while boiling. To prepare the highest quality agarose gels of any percentage, an additional 3-5 min of boiling after completely melting the agarose is recommended. A significant amount of water evaporates during this procedure and therefore restoring of the initial weight (in step 5) is required to obtain the desired percentage gel.
5. Weigh the flask again and if necessary, add hot distilled water to restore the initial weight.
   For high percentage gels (3-5%): check (by weighing) that the excess 10-20% of water has evaporated and, if needed, continue boiling to remove any excess, or add hot distilled water to restore the initial weight.
6. Cool the solution to 65-70°C. Pour carefully on a clean casting plate with an appropriate comb. If bubbles appear, push them carefully away to the sides with a pipette tip.
7. Solidify the gel for approximately 30 min before use.
8. Immerse the gel for at least one hour into the alkaline electrophoresis buffer (30 mM NaOH, 2 mM EDTA). Dilute 5 volumes of the DNA sample or ladder with one volume of 6X alkaline electrophoresis loading buffer (180 mM NaOH, 6 mM EDTA, 18% Ficoll 400, 0.05% bromcresol green).
9. Heat samples and ladder at 70°C for 5 min, then chill on ice for 3 minutes. Load onto the gel.
10. Run electrophoresis at 3 V/cm in alkaline electrophoresis buffer (30 mM NaOH, 2 mM EDTA) until the dye runs approximately two-thirds of the way down the gel.
    After electrophoresis the gel should be immersed for 30 min in 100-300 ml of 0.5 M Tris-HCl buffer, pH 7.5 and later stained in a 0.5 µg/ml ethidium bromide solution for 30 min. If staining is not enough, the whole procedure can be repeated.

1. For a nondenaturing 5% polyacrylamide gel solution of 40 ml, mix the following:

   10X TBE Buffer	4 ml
   20% acrylamide/bisacrylamide	10 ml
   Deionized water	26 ml

   Caution: acrylamide is a neurotoxin; always wear gloves, safety glasses, and a surgical mask when working with acrylamide powder.
2. Vigorously agitate the solution for 1 min by magnetic stirring to ensure complete mixing.
3. Add 48 µl of TEMED and swirl the flask to ensure that the solution is thoroughly mixed.
4. Immediately add 240 µl of fresh 10% (w/v) APS and mix thoroughly.
5. Pour the acrylamide between the gel plates and insert the comb.
   Clamp the comb in place at the top of the gel to avoid separation of the gel from the plates as the acrylamide polymerizes. Allow the gel to polymerize for 30 min.
   Important note: polymerization begins as soon as APS is added to the mixture, so all subsequent steps must be performed quickly.
6. After polymerization is complete, remove the comb and any bottom spacers from the gel. Wash the gel plates to clean any spilled acrylamide and be sure that the spacers are properly seated and clean. Fill the lower reservoir of the electrophoresis tank with 1X TBE buffer. Initially, place the gel into the lower tank at an angle to avoid the formation of air bubbles between the plates and the gel bottom. Clamp the gel plates to the top of the electrophoresis tank and fill the upper reservoir with 1X TBE so that the wells are covered.
7. Pre-run and warm the gel for at least 30 min at 5 V/cm (constant voltage).
   Load the recommended volume of the ladder, premixed with the appropriate electrophoresis loading dye solution. Use the same loading dye for the sample DNA.
8. Run the gel at 5 V/cm, taking care to avoid excessive heating. Run the gel for the time indicated in the certificate of analysis of the ladder.
9. Stain the gel in a 0.5 µg/ml ethidium bromide aqueous solution for about 30 min. Examine the gel under the UV light.

Note

1. For a denaturing 10% polyacrylamide gel solution of 40 ml, mix the following:

   10X TBE Buffer	4 ml
   20% acrylamide/bisacrylamide	10 ml
   UREA	19.2 g (to 8 M final concentration)
   Deionized water	to 40 ml

   Caution: acrylamide is a neurotoxin; always wear gloves, safety glasses, and a surgical mask when working with acrylamide powder.
2. Vigorously agitate the solution by magnetic stirring to ensure complete mixing and solving of UREA powder.
3. Add 40 µl TEMED and swirl the flask to ensure thorough mixing.
4. Immediately add 400 µl of fresh 10% (w/v) APS and mix thoroughly.
5. Pour the acrylamide between the gel plates and insert the comb.
6. Clamp the comb in place at the top of the gel to avoid separation of the gel from the plates as the acrylamide polymerizes. Allow the gel to polymerize for 30 min.
   Important note: polymerization begins as soon as APS is added to the mixture, so all succeeding actions must be performed promptly.
7. After polymerization is complete, remove the comb and any bottom spacers from the gel. Fill the lower reservoir of the electrophoresis tank with 1X TBE buffer. Initially, place the gel into the lower tank at an angle to avoid the formation of air bubbles between the plates and the gel bottom. Clamp the gel plates to the top of the electrophoresis tank and fill the upper reservoir with 1X TBE so that the wells are covered.
8. Pre-run and warm the gel for at least 30 min at 5 V/cm (constant voltage).
   

   Note

   Heat the gel (buffer) during the whole run at 60-70°C.
9. Wash the wells with 1X TBE buffer to remove UREA and gel pieces.
10. Load the samples.
11. Run the gel at 6 V/cm till the lower dye front reaches the three thirds of the gel.
12. Soak the gel for about 15 min in 1X TBE to remove the urea prior to staining.
13. Stain the gel in a 0.5 µg/ml ethidium bromide aqueous solution for about 30 min.
14. Examine the gel under the UV light.

• RNA ladders, as well as any RNA, are extremely sensitive to degradation by ribonucleases. Use only fresh electrophoresis buffers and freshly poured gels.
• Use clean electrophoresis chambers. For RNA gel analysis, avoid electrophoresis tanks used for DNA miniprep analysis since DNA minipreps may contain RNase A or T1.
• Use the same loading dye for samples and for RNA markers. 2X RNA Loading Dye is available separately and is provided with all RiboRuler™ RNA ladders. It contains ethidium bromide for RNA visualization on denaturing formaldehyde gels. If RNA fragments are separated on native agarose gels or on polyacrylamide/urea gels, additional staining with ethidium bromide is recommended.
• For native gels, add 0.5 µg/ml of ethidium bromide to the agarose gel and to the running buffer.

Note

1. Mix 1 volume of the 2X RNA Loading Dye and 1 volume of the RNA sample.
2. Heat at 70°C for 10 min.
3. Chill on ice for 3 minutes and spin down prior to loading on a gel.

Note

• Use an Erlenmeyer flask of at least three times larger volume than that of the solution to avoid boiling over.
• Use the same 1X electrophoresis buffer to prepare the gel and to run electrophoresis.
• Dilute 50X TAE or 10X TBE buffers to a 1X concentration immediately before use.
• Use TBE buffer for analysis of RNA bands smaller than 1500 b. For larger RNA, use TAE buffer.

1. Weigh out the required amount of agarose (depending on the gel %) into an Erlenmeyer flask.
2. Add the appropriate volume of either 1X TBE or 1X TAE buffer and swirl to mix.
3. Weigh the flask with the solution.
   For high percentage gels (3-5%): add an excess amount of distilled water to increase the weight by 10-20%.
4. Boil the mixture in a microwave oven (at middle power) until the agarose melts completely; swirl the flask several times while boiling. To prepare the highest quality agarose gels of any percentage, an additional 3-5 min of boiling after completely melting the agarose is recommended. A significant amount of water evaporates during this procedure and therefore restoring of the initial weight (in step 5) is required to obtain the desired percentage gel.
5. Weigh the flask again and if necessary, add hot distilled water to restore the initial weight.
   For high percentage gels (3-5%): check (by weighing) that the excess 10-20% of water has evaporated and, if needed, continue boiling to remove any excess, or add hot distilled water to restore the initial weight.
   Optional. For intensified gel staining add ethidium bromide to a final concentration of 0.5 µg/ml. Mix well and heat for an 1 minute without boiling.
6. Cool the solution to 65-70°C. Pour carefully on a clean casting plate with an appropriate comb. If bubbles appear, push them carefully away to the sides with a pipette tip.
7. Solidify the gel for approximately 30 min before use. Low percentage LM agarose gels can be solidified at 4°C.
8. Immerse the gel into the desired electrophoresis buffer.
9. Heat the RNA samples and ladder at 70°C for 10 min, then chill on ice for 3 min. Load onto the gel.
10. Run electrophoresis at 5 V/cm until the bromophenol blue runs approximately two-thirds of the way down the gel.

1. Freshly prepare 10X MOPS buffer: 0.4 M MOPS (pH 7.0), 0.1 M sodium acetate, 0.01 M EDTA (pH 8.0).
2. Prepare 1% TopVision™ Agarose gel as follows:
   
   • stir 1g of agarose powder in 72 ml of deionized water;
   • melt the agarose, and then add 10 ml of 10X MOPS buffer and mix;
   • when the agarose solution cools to 60°C, add 18 ml of fresh formaldehyde (37%) in a fume hood and mix thoroughly;
   • pour the gel.
3. Place the gel into an electrophoresis apparatus containing 1X MOPS buffer.
4. Heat the RNA samples and ladder at 70°C for 10 min, and then chill on ice for 3 min.
5. Load onto the gel.

Note

1. Prepare thick 1% TopVision™ Agarose gel in 0.01 M sodium phosphate buffer, pH 7.0.
2. Place the gel into an electrophoresis apparatus with 0.01 M sodium phosphate buffer, pH 7.0.
3. Prepare for loading 25 µl aliquots of the ladder/samples by adding:

   Glyoxal (40% solution)	4.5 µl
   DMSO	12.5 µl
   0.1 M sodium phosphate buffer, pH 7.0	2.5 µl

4. Mix and add:

   RiboRuler™ RNA Ladder or RNA sample	3 µl
   2X RNA Loading Dye	1 µl
   DEPC-treated Water	to 25 µl

5. Incubate for 1 hour at 50°C and then cool down to room temperature.
6. Load the samples on a gel.
7. Run electrophoresis at 5 V/cm until the bromophenol blue runs approximately two-thirds of the way down the gel.
8. Stain the gel in ethidium bromide solution (final concentration 0.5 µg/ml) in 0.5 M ammonium acetate for 15-30 min.
9. Wash the gel in fresh 0.5 M ammonium acetate solution for 15-30 min.

1. Prepare 20 ml of a 5% polyacrylamide gel containing 7 M urea by adding:

   47.5% acrylamide: 2.5% bis-acrylamide solution	2 ml
   10 M urea	14 ml
   10X TBE Buffer	2 ml
   10% freshly prepared ammonium persulfate	0.2 ml
   Deionized water	1.8 ml

2. Mix and add 10 µl TEMED. Mix again and pour the gel carefully avoiding the formation of air bubbles.
3. Insert the comb into the acrylamide and allow the gel to polymerize for at least 1 hour.
4. Fill the electrophoresis apparatus with 1X TBE buffer.
5. Heat the RNA samples and ladder at 70°C for 10 min, and chill on ice for 3 min.
6. Load onto the gel.
7. Run electrophoresis at 8 V/cm for about 1 hour.
8. Soak the gel for about 15 minutes in 1X TBE to remove urea prior to staining.
9. Stain the gel in 0.5 µg/ml ethidium bromide in 1X TBE solution for 15 min.

1. In each well, seed ~5x10⁴ adherent cells or ~5x10⁵ suspension cells 24 h prior to transfection.
   

   Note

   • The recommended confluency for adherent cells on the day of transfection is 50-70%.
   • Suspension cells should be in logarithmic growth phase at the time of transfection.
2. Dilute 1 µg of DNA in 100 µl of serum free DMEM or other growth medium.
3. Add 2 µl of TurboFect™ to the diluted DNA and mix the solution by pipetting.
4. Incubate 15-20 minutes at room temperature.
   

   Note

   • Prepare immediately prior to transfection.
   • We recommend starting with 1 µg of DNA and 2 µl of TurboFect™ per well in a 24-well plate (see Table below).
   • Subsequent optimization may further increase transfection efficiency depending on the cell line and transgene used.
5. Add 100 µl of the TurboFect™/DNA mixture drop-wise to each well. Do not remove the growth medium from the cells.
6. Gently rock the plate to achieve even distribution of the complexes.
7. Incubate at 37°C in a CO₂ incubator.
8. Analyze transgene expression 24-48 hours later.
   For stable transfection cells should be grown in selective medium for 10-15 days.
   Plates can be centrifuged for 2-5 min at 200xg to facilitate sedimentation of cells to the bottom of the plate.

Note

Note

• The recommended confluency for adherent cells on the day of transfection is 50-70%.

1. Dilute 1 µg of DNA in 100 µl of 150 mM NaCl. Vortex gently and centrifuge briefly.
2. Add 3.3 µl of ExGen 500 to the diluted DNA (not the reverse order) and vortex the solution immediately for 10 seconds.
3. Incubate for 10 minutes at room temperature.

Note

• We recommend starting with 1 µg of DNA and 3.3 µl (6 equivalents) of ExGen 500 per well of 24-well plate.
• Subsequent optimization may further increase the transfection efficiency depending on the cell line and transgene.

1. Add 100 µl of the ExGen 500/DNA mixture to each well.
   Generally, the volume of the ExGen 500/ DNA mixture represents 1/10 of the total volume of the culture medium.
2. Gently rock the plate to achieve even distribution of the complexes.
3. Centrifuge culture vessel, if possible, for 5 min at 280 x g.
4. Incubate at 37°C for 24-48 hours in a CO₂ incubator and analyse transgene expression.

Note

• The above incubation is designed for transfection without media change. If media change is preferred, incubate for 30 min (if centrifugation is possible) or for 3-4 hours (if centrifugation is not possible). Replace the media with the fresh complete growth media. Incubate for 24-48 hours.
• Transient expression of the transgene is monitored 24-48 hours post-transfection, while stable expression is monitored 10-15 days after transfection.

Note

1. Dilute 10 µg of DNA in 50 µl of a sterile 5% glucose solution. Vortex gently and centrifuge briefly.
2. Dilute 1.8 µl of ExGen 500 solution (6 equivalents) in 50 µl of sterile 5% glucose solution. Vortex gently and centrifuge briefly.
3. Add the diluted ExGen 500 to the diluted DNA (in this order). Vortex the solution immediately and spin down briefly.
4. Incubate for 10 minutes at room temperature.
5. Perform injections.
6. Monitor gene expression with the method most suitable for your studies.

Note

• The A₂₆₀/A₂₈₀ ratio should be at least 1.8 for purified DNA. It is important to use endotoxin-free DNA (less than 0.1EU/1 µg DNA).
• The amount of DNA and maximum injection volume depend on the experimental animal and the route of administration (see Tables 1 and 2 below) as well as on the targeted tissue or organ and on the expression vector.
• To prevent precipitation of the ExGen 500/DNA complex, the final concentration of DNA in the injection mix should not exceed 0.5 µg/µl.

Note

1. Wash the cells twice with cold 1X PBS buffer. Adhered cells can be washed in the transfection plates, suspension cells should be pelleted before washing.
2. Fix the cells with Fixation buffer for 10 minutes at room temperature while gently rocking the plate. Use 150 µl of the Fixation buffer for each well of a 24-well plate.
3. Wash the cells twice with cold 1X PBS buffer.
4. Stain the cells with freshly prepared Staining buffer for 2-20 hours at 37°C. Use 200 µl of Staining buffer for each well of a 24-well plate.
5. Count dark blue cells.

Note

• Reagents must be added in the order indicated.
• The TurboFect™/protein complexes should be prepared immediately prior to transfection.

1. In a 24-well plate, seed ~5x10⁴ adherent cells or ~1x10⁵ suspension cells per well 24 h prior to transfection.
   

   Note

   • The recommended confluency for adherent cells on the day of transfection is 70-90%.
   • Suspension cells should be plated at an optimal density ensuring logarithmic growth at the time of transfection.
2. Dilute 1 μg of protein in 100 μl of 0.15 M NaCl or serum-free growth medium.
3. Add 1 μl of Enhancer Solution and mix the solution by vortexing or pipetting.
4. Add 1 μl of TurboFect™ Protein Transfection Reagent and immediately mix the solution by vortexing or pipetting.
5. Incubate 15-20 min at room temperature.
6. Adherent cells: aspirate the growth medium. Suspension cells: centrifuge the cells at 200 x g for 5 min and aspirate the growth medium.
   Optional. Wash the cells once with serum-free growth medium.
7. Add 500 μl of serum-free growth medium to the cells.



Note

• Transfection can be performed in growth medium containing serum. In this case, the complete removal of the medium is not required. Aspirate half of the volume used for cell plating. Please note that the presence of serum will result in up to a 50% decrease in transfection efficiency.
8. Add 100 μl of the TurboFect™/protein complexes drop-wise to each well.
9. Gently rock the plate to achieve an even distribution of complexes.
10. Incubate for 2 h at 37°C in a CO₂ incubator.
11. Cells can be immediately used for subsequent experiments.
12. If cells are not used immediately, add 500 μl of complete growth medium with 2X serum and incubate until analysis. If toxicity is observed during prolonged incubation, remove the medium containing the TurboFect™/protein complexes 2 hours after transfection and add 1 ml of complete growth medium.
    

    Note

    • Prior to analysis, wash the cells thoroughly with serum-free growth medium (PBS or NaCl can also be used). This step removes undelivered protein.

1. Dilute 50 µg of DNA in 400 µl of a sterile 5% glucose solution. Vortex gently and centrifuge briefly.
2. Add 6 μl of TurboFect™ in vivo Transfection Reagent and mix the solution by pipetting.
3. Incubate for 15-20 min at room temperature.
4. Perform injections.
5. Monitor gene expression with the method most suitable for your studies.

Note

• The A₂₆₀/A₂₈₀ ratio should be at least 1.8 for purified DNA. It is important to use endotoxin-free DNA (less than 0.1EU/1 µg DNA).
• The amount of DNA and maximum injection volume depend on the experimental animal and the route of administration (see Tables 1 below) as well as on the targeted tissue or organ and on the expression vector.

Note

1. Seed ~5x10⁴ adherent cells or ~1x10⁵ suspension cells per well in 0.5 ml of growth medium 24 hours prior to transfection.

Note

The recommended confluency for adherent and suspension cells on the day of transfection is 70-90%.

2. Dilute 3 pmol of siRNA in 100 μl of 0.15 M NaCl or other serum-free medium for a final siRNA concentration of 5 nM in the cell culture.
3. Add 1 μl of TurboFect™ to the diluted siRNA and mix by pipetting.
4. Incubate 15-20 minutes at room temperature.

Note

The TurboFect™/siRNA complexes should be prepared immediately prior to transfection.

5. Add 100 μl of the TurboFect™/siRNA mixture drop-wise to each well.
6. Gently rock the plate to achieve an even distribution of the complexes.
7. Incubate at 37°C in a CO₂ incubator.
8. Assay gene suppression 24-72 hours later.

Note

1. Remove media from the cells or tissue.
2. Immerse cells/tissue in fresh staining buffer.
3. Incubate cells/tissue for 12-24 hours at 37°C.
4. Remove the staining buffer.
5. Wash with several changes of 50% ethanol (up to 12 hours per wash), until the cells/tissue clears.
6. Count dark blue cells.

Note

• Avoid exposure to light.
• Prepare fresh developing solution and use within an hour.
• BCIP-T and NBT solutions in dimethylformamide do not freeze. They are stable for approximately 2 years when stored at -20°C in the dark.

1. Prepare the following reaction mixture:

   5X reaction buffer for Terminal Deoxynucleotidyl Transferase	4 µl
   DNA fragments	1 pmol of 3'-ends
   (dATP or dTTP) or (dGTP or dCTP)	130 pmol or 60 pmol
   Terminal Deoxynucleotidyl Transferas	1.5 µl (30u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Incubate the mixture at 37°C for 15 min.
3. Stop the reaction by heating at 70°C for 10 min or by the addition of 2 µl 0.5 M EDTA.

Note

• Under the conditions described above, 100-130 dA or dT residues, or 20-30 dC or dG residues can be added per 3'-OH end of DNA.
• The efficiency of the reaction depends upon the type of 3'-OH termini of the DNA fragments. 3'-overhang ends are tailed with higher efficiency than recessed or blunt ends.

1. Add the following components to a reaction tube:
   

   Plasmid containing Bpu10I recognition sequence (20 µg)	20-356 µl
   10X Buffer R	40 µl
   Nb.Bpu10I	4 µl (20 u)
   Water, nuclease-free	to 400 µl

2. Vortex the tube and spin in a microcentrifuge for 3-5 seconds.
3. Incubate at 37°C for 1 hour.
4. Add Ѕ volume of phenol (200 µl) and Ѕ volume of chloroform/isoamyl alcohol (24:1) (200 µl), vortex for 10 seconds and centrifuge at 10,000 rpm for 5 minutes.
5. Transfer the upper aqueous phase to a fresh tube and add 1 volume (400 µl) of chloroform/isoamyl alcohol (24:1). Vortex and centrifuge for 5 minutes.
6. Repeat step 5 twice more.
7. Transfer the upper aqueous phase to a fresh tube. Add 1/10 volume of 3 M sodium acetate and 2.5 volumes of ice-cold ethanol. Mix and incubate at -20°C for 1 hour.
8. Centrifuge at 10,000 rpm for 10 minutes.
9. Pour off the supernatant and carefully wash the pellet with 200 µl of 75% ice-cold ethanol. Dry the pellet.
10. Dissolve DNA in 50 µl of water, nuclease-free.
11. Treat with Exonuclease III by adding the following components:
    

    10X reaction buffer for ExoIII	25 µl
    Exonuclease III	6 µl (1200 u)
    Water, nuclease-free	169 µl

12. Mix and incubate at 30°C for 10 min. Stop the reaction by heating at 70°C for 10 min.
13. Extract the reaction mixture with phenol/chloroform as described in steps 4-6, precipitate DNA as described in steps 7-9 and dissolve in 10-30 µl of deionized water. This solution should contain single-stranded DNA, suitable for DNA sequencing, site-specific mutagenesis, differential display, etc.

1. Add 15-20 u of RNase I per 1 µg of RNA. RNase I is ≥ 90% active within pH range 7.0-8.8 at salt concentration 100-200 mM. Incubation with RNase I can be performed simultaneously with the digestion of DNA by restriction endonucleases.
2. Incubate at 37°C for 30 minutes.
3. Purify DNA by spin column or phenol/chloroform extraction.