yeast_pcr

Yeast PCR

Set up

To begin, pull the plates of the strains you will be PCRing from the 4C fridge.

Open the MM PCR folder on the desktop.
Open the file called PCR Template.
Save as your initials, the strains you are testing, and the date.
Begin to fill out the spreadsheet with MM/DC numbers of the strains, the genotype, the mating type, etc.
Open the primers spreadsheet via the dropbox. Fill out the primer information for the respective primers that you are going to use. PROTIP: you can hold control to copy each of the cells you need and then control shift V to paste the information.
Decide the layout for both your boil and PCR. For each, you will need to test WT strains as well. For the PCR strips, you'll test each strain against WT and the master mix.
Depending on how many samples you are testing, you will need to calculate the amount of each reagent for the master mix. Keep in mind, you will want to make extra master mix for each strain. For example, one strain includes the strain itself, the two WT, and the blank = 9 * 4 = 36. It would be safe to round up to 40.

Boil Procedure

Make 1mL of .02 NaOH solution.
Put 10ul of the NaOH solution into each well that you will use for your boil.
Pick a small amount from each colony and resuspend in NaOH.
Mark your boil strips with sharpie over entire top and sides.
Incubate at 99C for 10 min. On the cycler you will press open method → yeast boil → start run.
Spin strips after boiling for 1-2 seconds.

PCR Procedure

Make your master mix.
Aliquot 9 ul of master mix to PCR tubes.
Transfer 1ul of boiled samples to master mix aliquots. Pull from supernatants, be careful not to disturb pellet.
Flick and swish.
RUN your PCR. You will need to check the TMs of each primer as well as the expected base pairs. Since you will have a forward and reverse primer with two different TMs, choose the lower one. Adjust the cycle accordingly.
Make sure you a labeled PCR rack on top of the cycler. Your samples will go into the mini fridge after they are done.