PCR Deletion Verification from Smash N Grab DNA

1. Do a Smash N Grab Yeast Genomic DNA Prep from the colonies of interest.

2. Sign up for a slot on one of the PCR Machines.

3. Set up PCR reactions in 8-Well PCR Strips. For each reaction:

Invitrogen PCR SuperMix, P/N 10572-014 22.5 ul

Forward primer from 100uM stock 0.25 ul

Reverse primer from 100uM stock 0.25 ul

Smash and Grab template DNA 1ul

ddH2O 1ul

4. Any extra of the master mix that you have, add in to any extra PCR wells you may have as a no template negative control. If you have all wells filled with master mix and samples, yet still have master mix left over, consider running the extra on another PCR strip if the quantity justifies using a whole strip and the time.

N.B.: Remember to completely thaw the supermix, forward and reverse primer!!!

N.B.: Write anealing temp on the PCR strips with a fine-tip Sharpie!!!

4. PCR Program, "VER" on Eppendorf machines, in Brett's folder or Mark's folder. I made a copy for Mark's folder because I changed the annealing step to a gradient so I can do several primers with different annealing teperatures at the same time in different strips.

N.B.: The Eppendorf PCR machines will not turn off when you open them and take your samples out! You have to find the cycler you were using on the menu screen (i.e., Cycler 1 or Cycler 2) and make sure you stop the program. Specifically, many programs have a 4C or 10C hold step at the end, and the machine will stay in that step unless you abort the program to end it, after you take your samples out.Edit