Described here is all information regarding polymerase chain reactions using Taq DNA polymerase, along with its optimization and troubleshooting. The standard reaction protocol and guidelines are largely based on NEB recommendations for Taq (2), and optimization information (additives, troubleshooting protocols) are 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 more error prone than high-fidelity polymerases (e.g. Q5) used to prepare DNAs for assembly/cloning. 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. –Shyam Bhakta
Reaction Setup
| Component | 10 µL Rxn | 25 µL Rxn | Final Concentration |
|---|---|---|---|
| 10× Standard Taq Reaction Buffer or 10× ThermoPol™ Reaction Buffer¹ | 1 µL | 2.5 µL | 1× |
| 10 mM dNTPs | 0.2 µL | 0.5 µL | 200 µM |
10 µM Forward Primer | 0.2 µL or 0.02 µL | 0.5 µL or 0.05 µL | 0.2 µM |
10 µM Reverse Primer | 0.2 µL | 0.5 µL | 0.2 µM |
| Template DNA/cells | variable 1 µL cells | variable | 10% V/V cell suspension |
| Taq DNA Polymerase | 0.0625 µL | 0.125 µL | 0.5% V / V with 5 U/µL enzyme. 0.025 U/µL rxn. |
| Nuclease-Free Water | to 10 µL | to 25 µL | - |
¹ ThermoPol Buffer contains a nonionic detergent to increase enzyme stability during longer incubations.
- Cell template preparation from colonies:
- Aliquot 20 µL PBS or growth medium (no antibiotics necessary) into as many 250 µL thermocycler tubes/strips as unique colonies you desire to run reactions on. Label with colony IDs.
- Using pipette tips, pick colonies into these tubes, wiping them on the inside of the tube into the liquid to dislodge the cells.
- You may optionally remove a small amount of cell suspension for later liquid or solid culturing, especially if wanting to pre-lyse the cells. A few µL may be multichannel pipetted onto an agar dish and grown for later use of isolates of desired genotypes. Similarly, a few µL may be used to inoculate liquid cultures for further experiments or DNA purification, accelerating growth by the ≥2 hr required for colony PCR and gel electrophoresis.
- Pre-lysis of cells may improve reliability of colony PCRs by removing cell solids and extracting DNA into the liquid. To do this, save some live cells for later growth if necessary (see previous step), before "boiling" the rest on a thermocycler in water, buffer, or growth medium at 98°C, 10 min and centrifuging down the cell debris to use the liquid cell extract supernatant as the PCR template.
- For as many colony PCRs, assemble reaction components without template cells on ice/cold block in 250 µL thermocycler tubes/tube strips (unless using a hot-start version of Taq which doesn't require keeping cold).
- Enzyme must be added after at least buffer and water are mixed.
- Make master mixes when possibles, as it reduces pipetting steps, reduces errors from pipetting small volumes, and maximizes component precision across reactions. Combine all common components for n reactions together and into reaction tubes aliquot reaction volumes less the volume of the variable component.
e.g., templates commonly vary across reactions, so for ten 10 µL reactions containing a combined volume of 1 cell template, combine water, buffer, dNTPs, primers, any enhancers, and polymerase; mix and aliquot 9 µL of this master mix across ten tubes, before adding their unique cell templates. Cell suspensions may be multichannel-pipetted into reactions for convenience.
- After adding last component, mix reaction with pipette or by closing, flicking, and centrifuging tubes to recollect liquid at bottom.
- Transfer the reactions to a thermocycler preheated to the denaturation temperature (95°C). Pausing just after starting the program will pause the protocol after the lid has finished (slowly) heating, after which pausing will pause the program with the block heated to the denaturation temp, step 1.
- Analyze PCRs using gel or capillary electrophoresis. As amplicon yields are ideally high, running smaller amounts of DNA than the entire reaction may give better DNA resolution.
Thermocycling
| Step | Temperature | Time | Notes | |
|---|---|---|---|---|
| Initial Denaturation | 95°C | 3 min | 2–5 min for complex templates, | |
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 | 60 s/kb | ||
| Final Extension | 68°C | 5 min | Holding temp is unnecessary and bad for thermocycler (1ref). |
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.
Troubleshooting
and adapt protocols using Taq
thermocycling times/temperatures.
Andrew's protocol
1. Pipette 8 µl of LB into PCR tubes and 5 ml into glass culture tubes.
- Saris, Per EJ, Lars G. Paulin, and Mathias Uhlén. "Direct amplication of DNA from colonies of Bacillus subtilis and Escherichia coli by the polymerase chain reaction." Journal of Microbiological Methods 11.2 (1990): 121-126. https://doi.org/10.1016/0167-7012(90)90012-U
- PCR Protocol for Taq DNA Polymerase | NEB