Guidelines for Preventing Contamination of PCR Reference Guidelines for Primer Design Estimation of Primer Melting Temperature

Transcription

1 Guidelines for Preventing Contamination of PCR During PCR more than 10 million copies of a template DNA are generated. Therefore, care must be taken to avoid contamination with other templates and amplicons that may be present in the laboratory environment. General recommendations to lower the risk of contamination are the following: 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, PCR frequently is contaminated by amplicons from previous PCR held in the same room. One of the most popular and efficient methods for prevention of carryover contamination is a use of uracil DNA glycosylase* (UDG) (1). A part or all of the dttp in the PCR reaction is substituted by dutp and therefore all PCR products generated in your working environment contain dutp. Prior to each PCR, short incubation with UDG eliminates such contaminating amplicons carried over from the previous PCR. Incorporation of dutp does not affect the intensity of ethidium bromide staining or the electrophoretic mobility of the PCR product, therefore the reactions can be analyzed by standard agarose gel electrophoresis. Taq DNA polymerase and all other non-proofreading polymerases will incorporate dutp into a PCR product, but proofreading polymerases or enzyme mixes containing such proofreading polymerases (e.g, Fermentas DreamTaq DNA Polymerase, High Fidelity Enzyme Mix or the Long PCR Enzyme Mix), do not incorporate dutp or may incorporate with much less efficiency. * Use of such enzyme in certain territories may be covered by patents and may require a license. Reference 1. Longo, M.C., et al., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions, Gene 93, 125-8, Guidelines for Primer Design Use the REviewer primer design software or follow general recommendations for PCR primer design below: PCR primers are generally 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. Estimation of Primer Melting Temperature For primers containing less than 25 nucleotides, the approx. melting temperature (Tm) can be calculated using the following equation:

2 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. Considerations for Subsequent Cloning of PCR Products 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. Components of the Reaction Mixture Template DNA Optimal amounts of template DNA in the 50 µl reaction volume are in the ng range for both plasmid and phage DNA, and in the µg range for genomic DNA. Higher amounts of template increase the risk of generation of nonspecific PCR products. Lower amounts of template reduce the accuracy of the amplification. All routine DNA purification methods are suitable for template preparation e.g., Genomic DNA Purification Kit (#K0512), GeneJET Plasmid Miniprep Kit (#K0502). Trace amounts of certain agents used for DNA purification, such as phenol, EDTA and proteinase K, can inhibit thermostable DNA polymerases. Ethanol precipitation and repeated washes of the DNA pellet with 70% ethanol normally remove trace contaminants from DNA samples. Primers The recommended concentration range of primers is µm. Too high primer concentrations increase the probability of mispriming and thereby appearance of nonspecific PCR products. For degenerate primers and primers used for long PCR higher primer concentrations in the range of µm are often favorable. Therefore start optimization from standard concentrations and increase if necessary. Mg 2+ Concentration Mg 2+ in general stabilizes primer-template complexes. PCR buffers for Taq DNA Polymerase are supplemented with Mg 2+, while in PCR with Pfu DNA Polymerase MgSO 4 is a preferable component. Due to the binding of Mg 2+ to dntps, primers and DNA templates, Mg 2+ concentration needs to be optimized for maximal PCR yield. The recommended concentration range is 1-4 mm. If the Mg 2+ concentration is too low, the yield of PCR product could be reduced. On the contrary, non-specific PCR products may appear and the PCR fidelity may be reduced if the Mg 2+ concentration is too high. If DNA samples contain EDTA or other metal chelators, the Mg 2+ ion concentration in the PCR mixture should be increased accordingly (1 molecule of EDTA binds 1 molecule of Mg 2+ (1)). Recommended Mg 2+ concentrations: Taq DNA Polymerase at Fermentas is supplied with two buffers: Taq buffer with KCl andtaq buffer with (NH 4 ) 2 SO 4. K + stabilizes primer annealing whereas NH 4 + has a destabilizing effect especially on weak hydrogen bonds between mismatched primertemplate base pairs. Therefore for standard PCR with Taq DNA Polymerase and 0.2 mm dntps the recommended MgCl 2 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 (NH 4 ) 2 SO 4. Due to antagonistic effects of NH 4 + and Mg 2+, Taq buffer with (NH 4 ) 2 SO 4 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 MgSO 4 is recommended. Volumes of 25 mm MgCl 2 or 25 mm MgSO 4 solutions required to reach a specific concentration of magnesium ions in the 50 µl reaction volume:

3 Final concentration, mm Volume of 25 mm MgCl 2 or MgSO 4, µl dntps The recommended concentration of each dntp is 0.2 mm. In certain PCR applications higher dntp concentrations are required. Due to the binding of Mg 2+ to dntps, Mg 2+ concentration needs to be adjusted accordingly. It is essential to have equal concentrations of all four nucleotides (datp, dctp, dgtp and dttp). If the nucleotide concentrations are not balanced, the PCR error rate may dramatically increase. Fermentas PureExtreme dntp Mixes contain either 2 mm or 10 mm, or 25 mm of each nucleotide. The concentrations of all four dntps are perfectly balanced to provide fidelity and to increase the yield of PCR products. To achieve 0.2 mm concentration of each dntp in the PCR mixture, use the following volumes of dntp Mixes: Volume of PCR mixture dntp Mix, 2 mm each (#R0241) dntp Mix, 10 mm each (#R0191) 50 µl 5 µl 1 µl 0.4 µl 25 µl 2.5 µl 0.5 µl 0.2 µl 20 µl 2 µl 0.4 µl 0.16 µl dntp Mix, 25 mm each (#R1121) To prepare 1 ml of working solutions of dntps (dntp Mixes) from individual 100 mm dntps or dntp Set, use the following volumes of reagents: Component dntp Mix, 2 mm each (#R0241) dntp Mix, 10 mm each (#R0191) datp, 100 mm 20 µl 100 µl 250 µl dttp, 100 mm 20 µl 100 µl 250 µl dgtp, 100 mm 20 µl 100 µl 250 µl dctp, 100 mm 20 µl 100 µl 250 µl Water, nuclease-free 920 µl 600 µl - Total volume 1 ml 1 ml 1 ml dntp Mix, 25 mm each (#R1121) Thermostabile DNA Polymerases Taq DNA Polymerase. Taq DNA polymerase is the most commonly used enzyme for PCR. It is suitable for most amplifcation reactions that do not require high fidelity enzyme or PCR products longer than 3 kb. Normally, u of Taq DNA Polymerase are recommended for a 50 µl volume of a PCR mixture. Nonspecific PCR products may appear at higher concentrations of the polymerase. However, it may be necessary to increase the amount of Taq DNA Polymerase to 2-3 u, if the PCR mixture contains inhibitors, for instance, due to contamination of the template DNA. Taq DNA polymerase, if PCR is assembled at room temperature, exhibits low but noticeable activity during the reaction set-up. As a result, non-specific priming events, such as mispriming or formation of primer dimers, which occur at ambient temperatures, will lead to generation of nonspecific amplification products during PCR. Therefore, PCR reaction set-up should always be performed on ice. DreamTaq DNA Polymerase. DreamTaq DNA Polymerase is an enhanced Taq DNA polymerase optimized for all standard PCR applications. It ensures higher sensitivity, longer PCR products and higher yields compared to conventional Taq DNA Polymerase. DreamTaq DNA Polymerase uses the same reaction set-up and cycling conditions as conventional Taq DNA Polymerase. An optimization of reaction conditions is generally not required. It is supplied with optimized DreamTaq buffer, which includes 20 mm MgCl 2. DreamTaq DNA Polymerase generates PCR products with 3 -da overhangs. PCR with DreamTaq DNA Polymerase is inhibited by dutp, but the enzyme can incorporate modified nucleotides.

4 Hot Start Taq DNA Polymerases. Hot start PCR uses enzymes, which have no activity at room temperature and are activated only at high temperatures during PCR cycling (e.g. TrueStart Hot Start Taq DNA Polymerase or Maxima Hot Start Taq DNA Polymerase). In hot start PCR non-specific amplification is reduced and target yield is increased. Using hot start DNA polymerases, PCR can be set-up at room temperature. TrueStart Hot Start TaqDNA polymerase has very short activation time (1 min) and can be used without changing of regular PCR cycling protocol. Maxima Hot Start Taq DNA Polymerase is activated in 4 min. Pfu DNA Polymerase. Pfu DNA Polymerase is a thermostable DNA polymerase with proofreading activity. It is one of the highest fidelity enzymes among thermostable DNA polymerases and is widely used in applications which require high fidelity amplification, e.g. cloning and expression. Normally, u of Pfu DNA Polymerase are used in a 50 µl volume of PCR mixture. The actual amount of enzyme required for optimal PCR yield and fidelity depends on the target to be amplified and on the presence of inhibitors in the PCR mixture. Pfu DNA polymerase is a slower enzyme than Taq DNA polymerase and it requires an elongation time of 2 min/kb. Also, Pfu DNA polymerase often requires more PCR cycles to produce sufficient amount of PCR product. Due to the intrinsic 3'=>5' exonuclease activity Pfu DNA polymerase should always be the last component added to the reaction mixture to avoid degradation of primers. It is also recommended to use longer PCR primers. Alternatively, phosphorothioate primers (exo- resistant primers) can be used to avoid primer degradation by Pfu DNA Polymerase (2). PCR Enzyme Mixes. Long PCR Enzyme Mix and High Fidelity Enzyme Mix are blends oftaq DNA Polymerase and a thermostable DNA polymerase with a proofreading activity. The two enzymes synergistically generate long PCR products in greater yields and higher fidelity than Taq DNA Polymerase alone. The Long PCR Enzyme Mix is also used for efficient amplification of GC-rich DNA regions. Normally, u of Enzyme Mix are used in a 50 µl volume of PCR mixture. Due to the 3'=>5' exonuclease activity of proofreading enzyme Enzyme Mixes should always be last components added to the reaction mixture to avoid degradation of primers. It is also recommended to use longer PCR primers. Alternatively, phosphorothioate primers (exo-resistant primers) can be used to avoid primer degradation by enzyme mixes. PCR Master Mixes. Thermostable DNA polymerases can be provided in a Master Mix format, a ready to use 2X concentrated solution, which includes DNA polymerase together with a PCR buffer and nucleotides. The Master mix is the most convenient and cost effective product for routine or high throughput PCR, where time for setting up a reaction and reproducibility of results are most important factors. Fermentas offers two PCR Master Mixes. The PCR Master Mix (2X) contains Taq DNA polymerase and is suitable for routine PCR. The PyroStart Fast PCR Master Mix (2X) contains a hot start Taq DNA polymerase and is formulated to work in fast thermal cycling conditions to reduce time not only dedicated to PCR set-up, but also to PCR cycling. PCR of less than 1 kb target can be completed in 25 min using this product. References 1. David, H., Modern Analytical Chemistry, Mc Graw Hill, 315, Skerra, A., Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity, Nucleic Acids Res., 20, , Cycling Parameters Amplification parameters greatly depend on the template, primers and parameters of the thermal cycler used. At Fermentas, functional PCR tests are performed on the GeneAmp PCR System 9700 (Applied Biosystems) or Mastercycler ep gradient S (Eppendorf). Initial DNA Denaturation. It is essential to completely denature the template DNA at the beginning of PCR to ensure efficient utilization of the template during the first amplification cycle. If GC content of the template is 50% or less, an initial 1-3 min denaturation at 95 C is sufficient. For GC-rich templates, this step has to be prolonged to 10 min. If longer initial denaturation step is required, or DNA is denatured at a

5 higher temperature, the Taq DNA Polymerase can be added after DNA is denatured to avoid a decrease in its activity. PfuDNA Polymerase can withstand a prolonged initial denaturation step due to its higher thermostability. Hot Start PCR. Hot start PCR uses Taq DNA polymerases which are inactive at room temperature and are activated during the initial DNA denaturation/ enzyme activation step at 95 C. TrueStart Hot Start Taq DNA polymerase has a very short activation time (1 min) and can be used without changing the regular PCR cyling protocol. Maxima Hot Start Taq DNA Polymerase activates in 4 min, therefore the initial denaturation step should be set to 4 min. Denaturation. Normally min DNA denaturation at C per cycle is sufficient. For GC-rich DNA templates, this step could be prolonged to 3-4 min. DNA denaturation can also be enhanced by the addition of either 10-15% glycerol or 10% DMSO, 5% formamide or M betaine. The melting temperature of the primertemplate complex decreases significantly in the presence of these reagents. Therefore, the annealing temperature has to be adjusted accordingly. Also, 10% DMSO and 5% formamide inhibit DNA polymerases by 50%. Thus, the amount of the enzyme should be increased, if these additives are used. Substitution of dgtp with 7-deaza-dGTP also can be used to decrease the melting temperature of PCR products. Primer Annealing. Annealing temperature should be 5 C lower than the lowest primer-template melting temperature (Tm). Annealing for min is normally sufficient. If nonspecific PCR products appear the annealing temperature should be optimized stepwise in 1-2 C increments. When additives are used which change the melting temperature of the primer-template complex (glycerol, DMSO, formamide and betaine), the annealing temperature also has to be adjusted. Touchdown PCR. Some experimental objectives may require PCR with a primer pair, which in conventional cycling protocol would generate unspecific products. In such cases a modification of conventional PCR, the touchdown PCR, may help to reduce the nonspecific amplification. In early PCR cycles an annealing temperature higher than the primer melting temperature is chosen and is decreased by 1 C every cycle or every second cycle until the desired or "touchdown" annealing temperature is reached. For the remaining cycles this touchdown temperature is used. By this the desired product is usually enriched over non-specific products. Extension. The rate of DNA synthesis by Taq DNA Polymerase and Pfu DNA Polymerase is highest at C. As a general rule, the extension step with Taq DNA Polymerase is 1 min at 72 C for PCR products up to 2 kb. For larger products, the extension time is prolonged by 1 min/kb. Since Pfu DNA Polymerase exhibits lower extension rate, an extension step of 2 min/kb at 72 C is recommended. Long PCR. For amplification of longer templates (>6 kb) a reduction of the extension temperature to 68 C is preferable to avoid enzyme loss during prolonged extension times. Number of Cycles. The number of cycles may vary depending on the amount of template DNA in the PCR mixture and the expected yield of PCR product. If less than 10 copies of the template are present in the reaction, about 40 cycles are required. With higher template amounts cycles are sufficient. Final Extension. After the last cycle, it is recommended to incubate the PCR mixture at 72 C for additional 5-15 min to fill-in the protruding ends of reaction products. If PCR product is to be cloned into TA vectors (for instance, using InsTAclone PCR Cloning Kit), the final extension step may be prolonged to 30 min to ensure the highest efficiency of 3'-dA tailing of PCR product. If PCR product generated with Taq DNA polymerase will be used for cloning using CloneJET PCR Cloning Kit the final extension step can be omitted. Fast PCR To drastically shorten the overall PCR cycling time Fermentas offers a specific formulation of hot start Taq DNA polymerase in convenient form of a master mix PyroStart Fast PCR Master Mix (2x). In Fast PCR the duration of all PCR cycling steps is reduced. The Primer Annealing and

6 Extension steps are combined in a single step of 25 s/kb with the possibility to optimize time down to 0 s. The Initial DNA Denaturation step is reduced to 60 s and the Final Extension step to 10 s. Therefore the overall time of a PCR <1 kb may be reduced to as little as 25 min compared to 2 h for conventional cycling protocols without compromising the yield, specificity and reproducibility. Standard PCR (reaction set up) The provided set-up is suitable for following Fermentas enzymes: DreamTaq DNA Polymerase, DreamTaq Green DNA Polymerase, Taq DNA Polymerase, recombinant andtaq DNA Polymerase, native. Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors, prepare a PCR master mix by adding water, buffer, dntps, primers andtaq DNA polymerase. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Aliquot the master mix into individual PCR tubes and add template DNA. 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 µm Reverse primer µm 25 mm MgCl 2 * 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 3. * Optional for PCR with DreamTaq DNA Polymerase, because the provided 10X DreamTaq buffer contains 20 mm MgCl 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. Recommended thermal cycling conditions: Step Temperature, C Time, min Number of Cycles Initial Denaturation Denaturation Annealing Tm Extension 72 1 min/kb Final Extension Hot Start PCR with TrueStart Hot Start Taq DNA Polymerase (reaction set up) Hot start PCR does not require reaction set up on ice. It involves an enzyme activation step in the cycling protocol. Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors, prepare a PCR master mix by adding water, buffer, dntps, primers andtaq DNA polymerase. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Aliquot the master mix into individual PCR tubes and add template DNA. Keep all reaction components on ice. Reaction set up can be performed at room temperature. 1. Gently vortex and briefly centrifuge all solutions after thawing.

7 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 25 mm MgCl µl Forward primer µm Reverse primer µm TrueStart Hot Start Taq DNA Polymerase u Water, nuclease-free to 50 µl Total volume 50 µl 5 µl (0.2 mm of each) 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. Recommended thermal cycling conditions: Step Initial Denaturation/Enzyme Activation Temperature, C Time, min Denaturation Annealing Tm Extension 72 1 min/kb Final Extension Number of Cycles Hot Start PCR with Maxima Hot Start Taq DNA Polymerase (reaction set up) Hot start PCR does not require reaction set up on ice. It involves an enzyme activation step in the cycling protocol. Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors, prepare a PCR master mix by adding water, buffer, dntps, primers andtaq DNA polymerase. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Aliquot the master mix into individual PCR tubes and add template DNA. Keep all reaction components on ice. Reaction set up can be performed at room temperature. 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 Forward primer µm Reverse primer µm 25 mm MgCl mm 5 µl (0.2 mm of each) Template DNA 10 pg 1 µg Maxima Hot Start Taq DNA Polymerase u Water, nuclease-free to 50 µl Total volume 50 µl 3. Gently vortex the samples and spin down to collect drops. Note 30

8 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. Recommended thermal cycling conditions: Step Initial Denaturation/Enzyme Activation Temperature, C Time, min Denaturation Annealing Tm Extension 72 1 min/kb Final Extension High Fidelity PCR (reaction set up) Number of Cycles Pfu DNA Polymerase is high fidelity DNA polymerase which has several differences fromtaq DNA polymerase. Pfu DNA polymerase should always be the last component added to the reaction mixture to avoid degradation of primers due to its intrinsic 3'=>5' exonuclease activity. Due to this feature, it is also recommended to use longer PCR primers. Pfu DNA polymerase is slower enzyme therefore for elongation step it is necessary to allow 2 min/kb. Also, Pfu DNA polymerase often requires more PCR cycles to produce sufficient amount of PCR product. Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors, prepare a PCR master mix by adding water, buffer, dntps, and primers. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Aliquot the master mix into individual PCR tubes and add template DNA 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 MgSO 4 5 µl dntp Mix, 2 mm each Forward primer µm Reverse primer µm 5 µl (0.2 mm of each) 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. Recommended thermal cycling conditions: Step Temperature, C Time, min Number of Cycles Initial Denaturation Denaturation Annealing Tm Extension 72 2 min/kb 35-40

9 Final Extension Long Range PCR (reaction set up) Fermentas offers two products suitable for long range PCR: Long PCR Enzyme Mix and High Fidelity PCR Enzyme Mix. The enzyme mixes contain Taq DNA polymerase and a thermostabile high fidelity DNA polymerase with proofreading activity. Due to the intrinsic 3'=>5' exonuclease activity of the high fidelity DNA polymerase, the enzyme mix should always be the last component added to the reaction mixture to avoid degradation of primers. Additionally, use of longer PCR primers (26-36 nt) is recommended. This protocol is provided for Long PCR Enzyme Mix. Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors, prepare a PCR master mix by adding water, buffer, dntps, and primers. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Aliquot the master mix into individual PCR tubes and add template DNA. 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 10X long PCR buffer with 15 mm MgCl 2 dntp Mix, 2 mm each PCR <30 kb 5 µl 5 µl 5 µl (0.2 mm of each) Forward primer µm µm Reverse primer µm µm PCR >30 kb and GC-rich PCR 5 µl (0.2 mm of each) Template DNA 10 pg 1 µg 1 ng 1 µg DMSO 2 µl (4%) Long PCR Enzyme Mix 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 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. Recommended thermal cycling conditions: Step Temperature, C Time Number of Cycles Initial Denaturation Denaturation s Annealing Tm-5 30 s Extension s/kb Denaturation s/kb Annealing Tm-5 30 s/kb Extension s/kb+5 s/cycle* Final Extension min * Recommended extensions to the elongation time: PCR fragment length, kb

10 Auto-extension per cycle, s RT-PCR Protocols