This protocol is intended for use with New England Biolabs' *Taq DNA Polymerase with ThermoPol® Buffer (M0267)*. The thermocycler parameters and master mix tables are provided for reference and should be transcribed into journal entries and filled with experimental values for each PCR performed. > [!warning] This page is for Taq polymerase! If you're looking for Q5 polymerase, click [[Polymerase chain reaction (Q5)|here]]! ## Setup 1. [ ] Determine the appropriate template and primers for your target. 2. [ ] Calculate the product size (bp). 3. [ ] Label PCR tubes for each reaction and control. 4. [ ] Prepare template DNA (purified DNA, gDNA, colony, plasmid, etc.) as needed. 5. [ ] Calculate master mix volumes, including 0.5 volumes worth of excess in your calculations.[^1] 6. [ ] Determine thermocycler parameters and input them into a thermocycler. ## Thermocycler parameters | \# of cycles | Step | Temperature (°C) | Time | | ------------ | ------------------------ | ---------------- | ------------ | | 1 | Initial denaturation[^2] | 95 | 30 s | | 30 | Cycling: Denaturation | 95 | 15 s | | 30 | Cycling: Annealing | *...varies* | 30 s | | 30 | Cycling: Extension | 68 | (1 min / kb) | | 1 | Final extension | 68 | 5 m | | 1 | Hold | 10 | ∞ | ## Master mix | Component | Vol. per 25 µL | Final concentration | | --------------------------------- | -------------- | ------------------- | | PCR water | *...to 25 µL* | — | | 10X ThermoPol reaction buffer[^3] | 2.5 µL | 1X | | 10 mM dNTPs | 0.5 µL | 200 µM | | 10 µM forward primer | 0.5 µL | 0.2 µM | | 10 µM reverse primer | 0.5 µL | 0.2 µM | | Template DNA[^4] | *...varies* | *...varies*[^5] | | Taq DNA polymerase | 0.125 µL | 0.025 U per µL | ## Protocol 1. [ ] Thaw all reagents on ice; perform all following steps on ice until directed otherwise. 2. [ ] Prepare the master mix by combining all components on ice except the template. Add the polymerase last. 3. [ ] Gently pipette up and down until all reagents are evenly mixed.[^6] 4. [ ] Distribute the master mix evenly across all PCR tubes.[^7][^8] 5. [ ] Add sterile water, instead of template, to the negative control tube. 6. [ ] Add the appropriate template DNA to all other reaction tubes. 7. [ ] If necessary, briefly centrifuge tubes to bring all liquid to the bottom. 8. [ ] Place tubes in the thermocycler and run the program. [^1]: For example, if you were preparing a PCR for 8 reactions, instead, prepare a master mix for 8.5 reactions. [^2]: For colony PCR, we have had success with 5 to 10 min initial denaturation periods before, if not doing any other prep work on the colony. Note that longer is not necessarily better; the shortest time you can get away with is usually best as longer denaturation goes on to damage the DNA. Results may vary. [^3]: When the reaction buffer is fully thawed, visually inspect it closely to look for any precipitated salts. If there are any, vortex the buffer until they are resuspended. Failure to do so can alter the concentration of both the present PCR and this mix, affecting all downstream PCRs. [^4]: If you are performing a colony PCR, review [[Protocols/Colony PCR|this guide]]. [^5]: Add template DNA to a final mass of 0.5 ng to 0.5 µg (gDNA) per 25 µL reaction or 0.5 pg to 5 ng (plasmid or viral DNA). [^6]: Be certain that the polymerase, in particular, is no longer visible. It is suspended in glycerol that will sink to the bottom of the tube when added. You must disturb that glycerol by this pipetting action until the glycerol is no longer visible when the bottom of the tube is disturbed. [^7]: When distributing your master mix, remember that you might not yet have 25 µL reactions at this stage because if you have not yet added your template. For example, if your template will be 0.5 µL of volume in each reaction and you have not yet added it, each aliquot you distribute should be only 24.5 µL at this step, not 25. [^8]: Optionally, if your template is the same in all reactions, you can save time by only removing the control reaction at this step. Add the sterile water to the control reaction as normal. Then, since you have not fractioned the rest of your master mix, you may add your total volume of the desired template directly to the remaining master mix, gently pipetting up and down before distributing the remaining master mix into the remaining reaction tubes.