Yeast Competent Cells - (Feb/06/2008 )
Yep, I do a 3 step with the GoTaq (95C/30sec;50C/30sec;72C/5min). I also start the reaction with 5 min at 95C to break open the cells. I use the GoTaq because that's what we have around the lab and its way cheaper than the advantage 2 mix (I save that for cDNA amplification). Give it a try! I wouldn't be surprised if other brands of plain old Taqs work, too.
I'm happy to help out! I know what it's like to have to figure this thing out by oneself.
my yeast pcrs are looking good now so thanks for your tips. I found I had to break open the cells with 15 min at 95C and I get good amplification with 3.5 min.
Many thanks, I owe you as beer !
Sorry to bother you with this again but im getting some variable results with my pcrs ! Have you found you need to PCR from cells fresh from the incubator or can you leave them in the fridge or on the bench for a couple of days. Also how big are the colonies you are touching with your tip ?
I haven't done anything special. I have done colony PCR off plates that have been at 4C for a week or so. But I have had a couple clones which were difficult to PCR, so I would drop the annealing temperature a couple of degrees and pick a different colony from the same plate. I had to test at least 3 colonies for one of my clones. I pick 'regular' sized colonies - like 2-3 mm. Also, I've noticed if I get too many cells in the tube it won't work. So make sure to touch the colony but not see any cells on the toothpick when you pull it out of the colony.
Best of Luck!
I seem to be having troubles with my transformation efficiencies, they were going really well but as I started to try and screen they dropped from around 4-5*10^5 to1*10^5 (with the pGBT9) whereas the protocol should expect 3*10^5. For the life of me I cannot trace the problem ! How are your efficiencies?