To prepare the reactions, you will first mix the primer pairs with a VERY SMALLnumber of yeast cells that you transfer from a colony to the tube with the tip of a P-20 or P-200 micropipette. The colony and primers will then be heated at 98 ̊C for 15 minutes to disrupt
the yeast cells. At that point, you will add an equal volume of a PCR master mix, containing nucleotides and the Taq polymerase, to each tube. The tubes will then be returned to the thermocycler for a typical PCR reaction.
1. Label the PCR tubes. The tubes are very small, so develop a code that you can use to identify the tubes. (Don’t forget to include the code in your notebook. The code should indicate which primers and strains are mixed in each tube.)
2. Prepare the primer mixtures. The final volume of the PCR reactions will be 20 μL. The primer mixture accounts for half the final volume, or 10 μL. The primers stock concentrations are 2.0 μM each. Pipette 5.0 μL each of the two primers that you would like to use into each PCR tube.What will the final concentration of each primer be in the actual PCR reaction?
Because of the extraordinary sensitivity of PCR reactions, it is very important not to cross-contaminate tubes with unintended primers. Change tips between every primer transfer.
3. Transfer a small quantity of yeast cells to each PCR tube. Lightly touch the tip of a P20 or P200 micropipette to a yeast colony. Twirl the micropipette tip in the tube containing your primer mix to release the cells. The most common error is transferring too many yeast cells, which will interfere with the PCR reaction. The right amount of yeast will fit on the tip of a pin.
4. Lyse the yeast cells. Place the tubes in the thermocycler for 15 min at 98 ̊C.
5. Set up the PCR reactions. Remove the tubes from the thermocycler and add 10 μL of PCR master mix to each tube.
6. Amplify the target gene sequences. Return the tubes to the thermocycler and start the PCR program.
Our thermocyclers are set for the following program:
- One cycle of denaturation: 95°C for 2 minutes
35 cycles of denaturation, annealing and extension:
95°C for 30 sec.
55°C for 30 sec.
72°C for 1 minute
- One cycle of extension: 72°C for 10 minutes