PCR Using Q5® High-Fidelity DNA Polymera (M0491)
/protocols/2012/09/27/pcr-using-q5-high-fidelity-dna-polymera-m0491
Protocol
1. Plea note that protocols with Q5® High-Fidelity DNA Polymera may differ from protocols with other polymeras. Conditions recommended below should be ud for optimal performance.
Reaction Setup:
We recommend asmbling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to u. Q5 High-Fidelity DNA Polymera may be diluted in 1X Q5 Reaction Buffer just prior to u in order to reduce pipetting errors.
狗能吃猫粮吗Notes: Gently 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:
Template:
U of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows:
1. Primers:
Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 can be ud to design or analyze primers. The best results are typically en when using each primer at a final concentration of 0.5 µM in the reaction.
2. Mg++ and additives:
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Mg++ concentration of 2.0 mM is optimal for most PCR products generated with Q5 High-Fidelity DNA Polymera. When ud at a final concentration of 1X, the Q5 Reaction Buffer provides the optimal Mg++concentration.
手机掉电快Amplification of some difficult targets, like GC-rich quences, 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 ud alone. It should be added only to reactions with the Q5 Reaction Buffer when other conditions have failed.
3. Deoxynucleotides: 斑石
The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. Q5 High-Fidelity DNA Polymera cannot incorporate dUTP and is not recommended for u with uracil-containing primers or templates.
4. Q5 High-Fidelity DNA Polymera concentration:
We generally recommend using Q5 High-Fidelity DNA Polymera at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the optimal concentration
of Q5 High-Fidelity DNA Polymera may vary from 10–40 units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and difficulty. Do not exceed 2 units/50 μl reaction, especially for amplicons longer than 5 kb.
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The 5X Q5 Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such as GC-rich templates or tho with condary 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 MgCl2 at the final (1X) concentration.
6. Denaturation:
An initial denaturation of 30 conds at 98°C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be ud (up to 3 minutes) for templates that require it.During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 cond denaturation at 98°C is recommended for most templates.
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7. Annealing:
Optimal annealing temperatures for Q5 High-Fidelity DNA Polymera tend to be higher than for other PCR polymeras. The NEB Tm Calculator should be ud to determine the annealing temperature when using this enzyme. Typically, u a 10–30 cond annealing step at 3°C above the Tm of the lower Tm primer. A temperature gradient can also be ud to optimize the annealing temperature for each primer pair.
For high Tm primer pairs, two-step cycling without a parate annealing step can be ud (e note 12). 伴君一生
8. 三年级口算题上册Extension:
The recommended extension temperature is 72°C. Extension times are generally 20–30 conds per kb for complex, genomic samples, but can be reduced to 10 conds per kb for simple templates (plasmid, E. coli, etc.) or complex templates < 1 kb. Extension time can be incread to 40 conds per kb for cDNA or long, complex templates, if necessary.A final extension of 2 minutes at 72°C is recommended.
9. Cycle number:
Generally, 25–35 cycles yield sufficient product. For genomic amplicons, 30-35 cycles are recommended.
10. 2-step PCR:
When primers with annealing temperatures ≥ 72°C are ud, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.
11. Amplification of long products:When amplifying products > 6 kb, it is often helpful to increa the extension time to 40–50 conds/kb.
