PCR Using Hot Start Taq DNA Polymerase

PCR Using Hot Start Taq DNA Polymerase (M0495) PCR The following guidelines are provided to ensure successful PCR using New England Biolabs' Hot Start Taq DNA Polymerase. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations, or amplicons greater than 5 kb may require further optimization. Reaction setup: Due to the hot start nature of the enzyme, reactions can be assembled on the bench at room temperature and transferred to a thermocycler. No separate activation step is required to release the inhibitor from the enzyme. Add to a sterile thin-walled PCR tube: Component 25 µl reaction 50µl reaction Final Concentration 10X Standard Taq Reaction Buffer 2.5 μι 5 μι 1X 10 mM dNTPs 0.5 μι ابر 1 200 μΜ 10 µM Forward Primer 0.5 μι 1 μι 0.2 μΜ (0.05-1 µM) 10 µM Reverse Primer 0.5 μι 1 με 0.2 μΜ (0.05-1 μΜ) Hot Start Taq DNA Polymerase 0.125 μι 0.25 μι 1.25 units/50 μι PCR Template DNA variable variable < 1,000 ng Nuclease-free water to 25 μι το 50 μι Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Transfer PCR tubes to a PCR machine and begin thermocycling: Thermocycling conditions for a routine PCR: Initial Denaturation 30. Cycles STEP TEMP TIME 95°C 30 seconds 95°C 45-68°C 68°C 15-30 seconds 15-60 seconds 1 minute/kb Final Extension 68 deg * C 5 minutes Hold 4-10°C General Guidelines: 1. Template: Use 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: DNA Genomic Amount Plasmid or viral 1 ng-1 µg 1 pg-1 ng 2. Primers: Oligonucleotide primers are generally 20-40 nucleotides in length and ideally have a GC content of 40-60%. Computer programs such as PrimerSelect™ and Primer3 can be used to design or analyze primers. The final concentration of each primer in a PCR may be 0.05-1 µM, typically 0.1-0.5 μΜ. 3. Mg and additives: M g^ ++ concentration of 1.5-2.0 mM is optimal for most PCR products generated with Hot Start Taq DNA Polymerase. The final M g^ ++ concentration in 1X Standard Taq Reaction Buffer is 1.5 mM. This supports satisfactory amplification of most amplicons. However, M g^ ++ can be further optimized in 0.5 or 1.0 mM increments using MgCl2 (NEB# 89021). Amplification of some difficult targets, like GCrich sequences, may be improved with additives, such as DMSO (1) or formamide (2). 4. Deoxynucleotides: The final concentration of dNTPs is typically 200 µM of each deoxynucleotide. 5. Hot Start Taq DNA Polymerase Concentration: We generally recommend using Hot Start Taq DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 µl reaction). However, the optimal concentration of Hot Start Taq DNA Polymerase may range from 5-50 units/ml (0,25-2,5 units/50 µl reaction) in specialized applications, 6. Denaturation: No separate activation step is required to release the hot start inhibitor from the enzyme. An initial denaturation of 30 seconds at 95 deg * C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GCrich sequences, a longer denaturation of 2-4 minutes at 95 deg * C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute incubation at 95 deg * C recommended to lyse cells. During thermocycling a 15-30 second denaturation at 95 deg * C is recommended. 7. Annealing: The annealing step is typically 15-60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45 - 68 deg * C Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5 deg * C the calculated Tm. We recommend using NEB's Tm Calculator to determine appropriate annealing temperatures for PCR. When primers with annealing temperatures above 68 deg * C are used, a 2-step PCR protocol is possible (see #10). 8. Extension: The recommended extension temperature is 68 deg * C Extension times are generally 1 minute per kb. A final 9. Cycle number: Generally, 25-35 cycles yield sufficient product, Up to 45 cycles may be required to detect low-copy-number targets. 10. 2-step PCR: When primers with annealing temperatures above 68°C are used, a 2-step thermocycling protocol is possible. Thermocycling Conditions for a Routine 2-Step PCR: STEP Initial Denaturation 30 Cycles Final Extension Hold TEMP 95°C 30 seconds 95°C 68°C 68°C 4-10°C TIME 15-30 seconds 1 minute/kb 5 minutes 11. PCR product. The PCR products generated using Hot Start Taq DNA Polymerase contain dA overhangs at the 3' end; therefore the PCR products can be ligated to dT/dU-overhang vectors. References: 1. Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374. 2. Sarkar, G., Kapelner, S. and Sommer, S.S. (1990). Nucleic Acids Res.. 18, 7465.