PCR protocol for amplification of MLST genes
1) For each 1005l PCR, use the following reaction components (multiply these volumes by the number of PCR reactions + 1 to make up a single master mix which can then be dispensed in 99.05l amounts into numbered, thin-walled PCR tubes):
| Reagent | Volume | Notes |
|---|---|---|
| H2O | 46.5µl | |
| 10x reaction buffer | 10.0µl | |
| PCR primer 1 | 10.0µl | [10µM primer stocks = 1µM final conc.] |
| PCR primer 2 | 10.0µl | |
| MgCl2 | 6.0µl | [25mM stock = 1.5mM final conc.: needs to be optimised for other primer sets] |
| dNTP mix | 16.0µl | [125µl of each 10mM dNTP + 500µl H2O = working stock] |
| Taq polymerase | 0.5µl | [2.5units of enzyme] |
| Total | 99.0µl |
2) Add 1µl of the appropriate template DNA (approximately 50ng/µl) to each tube. Remember to set up a negative PCR control, which consists of reaction components and NO added template DNA. If you are optimising the PCR, using new template DNA or using a new set of primers, it is advisable to also set up a positive PCR control by including a reaction that contains template DNA which has been amplified reliably in previous PCRs.
3) Place the tubes into the thermal cycler and close the lid, ensuring that the heated plate inside the lid is in contact with the tops of the tubes, by rotating the screw on the top of the lid until tight.
4) Cycling conditions:
We use the following program:
| Step | Temperature /0C | Time /min |
|---|---|---|
| 1 | 94 | 2 |
| 2 | 94 | 1 |
| 3 | 55-60 | 1 |
| 4 | 72 | 2 |
| 5 | repeat steps 2, 3 and 4 a further 29 times | |
| 6 | 72 | 2 |
| 7 | 4 | forever |
The annealing temperature should be determined for each different set of primers, but primers are generally designed to have optimal annealing temperatures of 55-600C (Tm values >600C).
5) After cycling, check that amplification was successful by running 55l of each reaction on an agarose gel, with size standards.
6) Purification of PCR products:
Transfer the contents of each PCR tube into labelled 1.5ml Eppendorf tubes. Add 60µl of 20% PEG 8000/ 2.5M NaCl to each tube and mix. Incubate for 15 min at 370C. Pellet the PCR products by spinning in a centrifuge at maximum speed for 10 minutes. Discard the supernatant and wash the DNA pellet by adding 0.5ml of 70% EtOH and spin at maximum speed for a furher 5 min. Discard the supernatant and dry pellets in the vacuum dryer (medium rate).
7) Resuspend the dried PCR products in 20-30 µl sterile H2O. Store at -200C.