Crimson Taq DNA Polymerase General Guidelines
- Reaction setup:
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (94-95°C).
Using high quality, purified DNA templates greatly enhances the success of PCR reactions. Recommended amounts of DNA template for a 50 μl reaction are as follows:
genomic DNA 1 ng-1 µg
plasmid or viral DNA 1 pg-1 ng
Primers are generally 20-40 nucleotides in length and ideally have a GC content of 40-60% (2). Computer programs such as Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) can be used to design or analyze primers. The final concentration of each primer in a typical PCR reaction is 0.05-1 μM, ideally 0.2-0.5 μM.
- Mg++ and additives:
The final Mg++ concentration in 1X Crimson Taq Reaction Buffer is 1.5 mM. This gives satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.5 or 1.0 mM increments using MgCl2.
For some difficult targets such as GC-rich sequences, additives such as DMSO (3) and formamide (4) may be included to improve amplification.
An initial denaturation of 30 seconds at 95°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, longer denaturation of 2-4 minutes at 95°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 95°C is recommended. Subsequent denaturation can be done between 10-30 seconds.
The annealing step is typically 15-60 seconds. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm.
Extensions are recommended to be carried out at 68°C. A final extension of 5 minutes at 68°C is recommended.
- 2-step PCR
When primers with annealing temperatures above 60°C are used, a 2-step PCR protocol is possible.
Initial denaturation: 95°C 30 seconds
25-45 cycles: 95°C 15-30 seconds; 60-68°C 1 minute per kb;
Final extension: 60-68°C 5 minutes
Final soak: 4-10°C
- Cycle number:
Generally, 25-35 cycles give optimal amplification. Up to 45 cycles may be required to detect low-copy targets.
- PCR product:
The PCR products generated using Crimson Taq DNA Polymerase contain dA overhangs at the 3´-end; therefore, the PCR products can be ligated to dT/dU-containing vectors.