The standard Taq DNA polymerase chain reaction protocol and guidelines are largely based on NEB recommendations for NEB Taq (2), and Thermo recommendations for DreamTaq Green 2× PCR master mix (3); optimization information (additives, troubleshooting protocols) is from elsewhere. Colony PCR was first described by Saris et al., 1990 (1).
Genotyping PCRs are PCRs intending to produce products whose presence and size are used to infer information about the genotype of the sample. A colony PCR is a genotyping PCR that uses cells as the template. Taq DNA polymerase is far, far more error prone than high-fidelity polymerases used to prepare DNAs for assembly/cloning (e.g. 5000× less than Q5). However, Taq is quite more economical to use, and for genotyping PCRs, sequence fidelity is generally not needed, as PCR products are not intended for downstream use. Additionally, Taq polymerase is robust enough to handle a sizeable amount of bacterial colonies or suspensions being added directly to the PCR unpurified, simplifying genotyping. Colonies can be resuspended in a single PCR to use it as a template, followed by inoculating broth or agar with the residual cells on the tip; or colonies can first be resuspended in liquid and a portion of the resuspension used in a PCR (required for using one colony/suspension in multiple PCRs).
PCR master mixes combine the enzyme, buffer, and dNTPs at a 2× concentration, so that only primers, template, and remaining water need be added. Some mixes are formulated for GC-rich templates. Some enzymes or PCR mixes have a blend of DNA polymerases for robustness, e.g. NEB OneTaq, which NEB has compared with other manufacturers' products against high-GC templates. Some polymerases or mixes have hot-start capability, which allow room-temp reaction setup without worry of nonspecific amplification, through an aptamer keeping the polymerase inactivated until the reaction is heated. Some mixes (ones with a color) already have a density reagent and tracking dyes as found in gel loading dyes, and can thus be loaded directly into a gel after thermocycling, e.g. NEB "quick-load" products or ones with colors in their name.
–Shyam Bhakta
Component | 10 µL Rxn | 25 µL Rxn | Final Conc |
---|---|---|---|
Nuclease-free water | to 10 µL | to 25 µL | – |
10× Standard Taq Reaction Buffer or 10× ThermoPol™ Reaction Buffer¹ | 1 µL | 2.5 µL | 1×. Mix with water before adding enzyme. |
Taq DNA polymerase | 0.0625 µL | 0.125 µL | 0.5%V/V with 5 U/µL enzyme. 0.025 U/µL rxn. |
10 mM dNTPs | 0.2 µL | 0.5 µL | 200 µM |
10 µM forward primer or 100 µM | 0.2 µL or 0.02 µL | 0.5 µL or 0.05 µL | 0.2 µM each |
10 µM reverse primer or 100 µM | |||
Template DNA/cells | variable 1 µL cells | variable 2.5 µL cells | 10%V/V cell suspension 1 pg–1 ng plasmid/viral DNA 1–1000 ng genomic DNA |
¹ThermoPol Buffer contains a nonionic detergent to increase enzyme stability during longer incubations.
Component | 10 µL Rxn | 25 µL Rxn | Final Conc |
---|---|---|---|
2× PCR master mix | 5 µL | 12.5 µL | Contains polymerase, dNTPs, and buffer. |
Nuclease-free water | 5 µL – x | 12.5 µL – x | x = Σ primers + template |
10 µM forward primer or 100 µM | 0.1–1 µL or 0.01-0.1 µL | 0.25–2.5 µL or 0.025–0.25 µL | 0.1–1 µM each |
10 µM reverse primer or 100 µM | |||
Template DNA/cells | variable, 1 µL cells | variable, 2.5 µL cells | 10%V/V cell suspension 0.01–1 ng plasmid/viral DNA 100–1000 ng genomic DNA |
Step | Temperature | Time | Notes | |
---|---|---|---|---|
Lid preheating | 105°C | |||
Initial Denaturation | 95°C | 1–3 min | 1 min for purified plasmid/linear/bacterial DNA. | |
25–35 30–35 | Denaturation | 95°C | 15 s | 15–30 s. |
Annealing | *50–68°C | 30 s | 15–60 s. *Find Tanl on Tm calculator | |
Extension | 68°C | 1 min/kb | ||
Final Extension | 68°C | 5 min | Holding temp is unnecessary and bad for thermocycler (ref). |
Note: When needing to destroy non-synthetic (non-amplicon) DNA, DpnI will not work in standard Taq buffer, requiring gel/column purification before digesting. DpnI does have full activity in ThermoPol reaction buffer, however.
Step | Temperature | Time | Notes | |
---|---|---|---|---|
Lid preheating | 105°C | |||
Initial Denaturation, Enzyme Activation | 95°C | 1–3 min | 1 min for purified plasmid/linear/bacterial DNA. | |
25–35 40 cycles: | Denaturation | 95°C | 30 s | 3–4 min for high-GC. |
Annealing | *50–68°C | 30 s | Tm – 5°. *Find Tanl. | |
Extension | 72°C | ≥1 min | 1 min for ≤2 kb; for >2 kb add 1 min/kb. Reduce to 68° when >6 kb. | |
Final Extension | 72°C | 5–15 min | Holding temp is unnecessary and bad for thermocycler (ref). |
See Q5/Phusion PCR page's PCR additives and Troubleshooting sections, and first adapt the concepts of ramped or gradient Tanl while keeping other thermocycling parameters in accordance with manufacturer recommendations.