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1、PCR Using Q5 High-Fidelity DNA Polymerase (M0491)https:/ Please note that protocols with Q5 High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.Reaction Setup:We recommend assembling all reaction component
2、s on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98C). All components should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors.Notes: Gently
3、mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid. Transfer PCR tubes to a PCR machine and begin thermocycling.Thermocycling Conditions for a Routine PCR:General Guidelines:Temp
4、late:Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 l reaction are as follows:1. Primers:Oligonucleotide primers are generally 2040 nucleotides in length and ideally have a GC content of 4060%. Computer programs such asPr
5、imer3can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 M in the reaction.2. Mg+and additives:Mg+concentration of 2.0 mM is optimal for most PCR products generated with Q5 High-Fidelity DNA Polymerase. When used at a f
6、inal concentration of 1X, the Q5 Reaction Buffer provides the optimal Mg+concentration.Amplification of some difficult targets, like GC-rich sequences, may be improved by the addition of 1X Q5 High GC Enhancer. The Q5 High GC Enhancer is not a buffer and should not be used alone. It should be added
7、only to reactions with the Q5 Reaction Buffer when other conditions have failed.3. Deoxynucleotides:The final concentration of dNTPs is typically 200 M of each deoxynucleotide. Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or te
8、mplates.4. Q5 High-Fidelity DNA Polymerase concentration:We generally recommend using Q5 High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 l reaction). However, the optimal concentration of Q5 High-Fidelity DNA Polymerase may vary from 1040 units/ml (0.52 units/50 l r
9、eaction) depending on amplicon length and difficulty. Do not exceed 2 units/50 l reaction, especially for amplicons longer than 5 kb.5. Buffers:The 5X Q5 Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicon
10、s, such as GC-rich templates or those with secondary structure, the addition of the Q5 High GC Enhancer can improve reaction performance. The 5X Q5 Reaction Buffer is detergent-free and contains 2.0 mM MgCl2at the final (1X) concentration.6. Denaturation:An initial denaturation of 30 seconds at 98C
11、is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it.During thermocycling, the denaturation step should be kept to a minimum. Typically, a 510 second denaturation at 98C is recommended for most templates.7. An
12、nealing:Optimal annealing temperatures for Q5 High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. TheNEB TmCalculatorshould be used to determine the annealing temperature when using this enzyme. Typically, use a 1030 second annealing step at 3C above the Tmof the lower Tmp
13、rimer. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.For high Tmprimer pairs, two-step cycling without a separate annealing step can be used (see note 12).8. Extension:The recommended extension temperature is 72C. Extension times are generally 203
14、0 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid,E. coli, etc.) or complex templates 6 kb, it is often helpful to increase the extension time to 4050 seconds/kb.12. PCR product:The PCR products generated using Q5 High-Fidelity DNA Polymerase have blunt ends. If cloning is the next step, then blunt-end clonin
