Dear all,
we are working with Yarrowia lipolytica and we have to screen quite some colonies for integration of different genes. We tried the zymolyase protocol to break up the cells and also a quick boiling (10-15s) of the colonies resuspended in PCR mastermix in microwave, which both worked well for S.cerevisiae, but not for Y.lipolytica - the results are inconsistent, sometimes it works, and sometimes it just does not.
Do you have any good alternative for Y.lipolytica, since we are doing the gDNA extractions at the moment which requires a lot of resources and time.
Thank you very much!
Martin
Hi,
I used for another yeast species (more like cerevisiae though) a modified s.cerevisiae protocol: break cells with zymolase, boil for 1min with 0.1% SDS, spin everything down and use supernatant for PCR (no more the 10% of the PCR reaction volume).
Aditionally I tried also a 45 min in 50mM NaOH + 0,5% SDS, which also did not give proper results. I plan a bid bigger study to try different things and I will include your protocol as well. Thank you!
Hi Martin, I’m PCRing science out of Yarrowia using the following protocol:
- pick colony with a pipette tip and resuspend in 5 µL or more of 20 mM NaOH (5 µL x n depending on how many different PCRs you want to run with one single clone)
- streak out the tip on a master plate, otherwise the clone will be gone
- cover the cell suspension with two drops of paraffin oil or seal with a lid
- boil for 10 min e.g. in a PCR cycler
- add 5 µL of the lysate into 45 µL standard PCR master mix containing everything but a template (I agree with Eimantas to not use more than 10% of template solution)
- run PCR according to the desired fragment size and used polymerase
This protocol works so far very reliable with different polymerases (Taq, OptiTaq and Q5) and delivers, depending on the primers, PCR products up to a couple of kbs.
Amplifying Regards
Thomas
- Badges
- Science method