Taq DNA Polymerase is the enzyme most widely used in the Polymerase Chain Reaction (PCR). The following guidelines are provided to ensure successful PCR using New England Biolabs' Taq 2X Master Mix. These guidelines cover routine PCR reactions. PCR of templates with high GC content, high secondary structure, low template concentrations or which produce products greater than 5 kb may require adaptations of these conditions.
- DNA Template: Using high quality, purified DNA templates greatly enhances the success of PCR reactions. It is also critical that DNAcontamination from previous related PCR reactions be avoided.
For typical detection, approximately 104 copies of the target DNA are required to detect a product in 25-30 cycles of PCR. Typically, this is 0.1-1 ng/ml final concentration of plasmid or viral templates, and 1-10 μg/ml of genomic templates. Use a colony less than 1 mm2 in size and resuspend completely before cycling.
- frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgiPrimers: Oligonucleotide primers are generally 20-30 nucleotides in length and ideally have a GC content of 40-60%, with GC residues spaced evenly within the primer. Calculated melting temperatures (Tm) for the two primers should generally be from 42-65°C and within 5°C of each other.
We recommend designing primers for PCR with the aid of a primer design computer program to achieve best results, such as PrimerSelect™ (DNAStar Inc, Madison, MI) and Primer3 (http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi).
The final concentration of each primer in a typical PCR reaction is 0.1-0.5 μM, ideally 0.2 μM.
- Magnesium Concentration: The magnesium concentration in the Taq 2X Master Mix is 3 mM, which gives a final 1.5 mM in PCR reactions. This results in satisfactory amplification of most amplicons. However, magnesium can be further optimized in 0.5 mM increments using MgCl2.
- Taq 2X Master Mix Concentration: Add Taq 2X Master Mix to a final concentration of 1X.
- Starting Reactions: Nonspecific primed synthesis during reaction setup and first heating cycle has been identified as a source of undesired products in some PCR reactions. This can often be avoided by assembling all reaction components on ice, adding the Taq 2X Master Mix last and immediately transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).
- Denaturation Temperature and Duration: An initial denaturation at 95°C is required prior to PCR cycling to fully denaturate the DNA. An initial 5 minute denaturation at 95°C is recommended for colony PCR. It is important to resuspend the colony completely.
During thermocycling, a 15 second-one minute denaturation at 94°C-95°C should be utilized, although this can depend on the thermocycler and tubes used. Consult the product literature accompanying the thermocycler being used for more specific recommendations.
- Annealing Temperature and Duration: Annealing temperatures should be chosen to match the Tm values of the primer pair, and are typically between 45°C-70°C. Annealing times of 15 seconds to one minute are usually adequate. Annealing temperature can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm.
- Extension Time: Extensions are recommended to be done at 68°C. As a general rule, extension times of 1 minute per 1000 base pairs should be employed. For products less than 1 kb, an extension time of 45-60 seconds should be used. A final extension of 5 minutes at 68°C is recommended.
- Cycling Conditions: A typical PCR reaction is shown:
1 cycle: 94°C-95°C 1-5 minutes
25-40 cycles: 94°C-95°C 15-30 seconds; 45°C-70°C 10-30 seconds; 68°C 1 minute per kb
1 cycle: 68°C 5 minutes (to finish replication on all templates)
1 cycle: 4°C-10°C indefinite period (storing the sample prior to further analysis)