PCR protocol for amplification of MLST genes

1) For each 50µl 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 49µl amounts into numbered, thin-walled PCR tubes):

10x reaction buffer
Reagent Volume Notes
H2O 40.75µl  
PCR primer 1 1µl [10µM primer stocks = 1µM final conc.]
PCR primer 2 1µl  
dNTP mix 1µl [10mM stock]
Taq polymerase 0.25µl [5 units/µl stock]
Total 49µl  

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 1
4 72 1
5 repeat steps 2, 3 and 4 a further 34 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 55-60°C (Tm values >60°C).

5) After cycling, check that amplification was successful by running 5µl 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 37°C or 30 min at room temperature. 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 -20°C.