Hi, I'm trying to transform S. cerevisiae with a 11Kb plasmid. I use the LiAc method, and this has always worked for me in the past. Recently however, I have been unable to get any transformants on my selective plate. I've tested every step of the protocol and nothing seems to be wrong, but I still can't get it to work. Could someone please help?
Are you increasing the amount of plasmid DNA you're using? If you normally use 100-200ng of 5Kb plasmid, then for an 11Kb plasmid you'd need 200-400ng to get an equivalent amount of plasmid (it solved my problems at least).
Hi Swathi,
you could try electroporation of your yeast cells. Sometimes, this results in better transformation efficiency. Maybe the protocol from the article shown in the link can help.
Hi John, I usually use around 500ng of DNA for my transformation. I've been using the same amount for the ones that haven't worked.
Katrin- I've tried electroporation, but I've had no colonies on the plate. I've changed the competent cells, the DNA, I make fresh PEG each time, and I even bought a new batch of salmon sperm DNA. Nothing seems to be working.
Couple of possibilities come to my mind:
1) Are you using the correct PEG, 3350? Is it freshly made?
2) Are you using the correct drop-out/drug plates?
3) If the plasmid has a drug-resistance marker, are you giving cells time to recover on no-drug media?
4) Are you sure your plasmid is OK? I used to work with a really long plasmid that got rearranged in E. coli and lost some of the yeast elements.
Hope this helps!
Hi Swathi,
you could try the transformation with a plasmid you know has worked in the past, if you get colonies probably the issue is with your 11 kb plasmid and not your reagents. Did you shear the salmon sperm dna? There are high efficiency LiAc protocols in which you use fresh log phase cells to get a higher transformation efficiency, you could try this. Also double check the selective marker on your plasmid. Good luck!
I have also same kind of problem from last 6 months. I tried a lot, I have also double check each and every step. Likewise I'm trying to transform S. cerevisiae with a SsDNA and my gene of interest and Iam using the same LiAc method but still trying..could not find out the troubleshoot.
You might want to (1) increase the plasmid DNA concentration to 2-4 ugs per transformation (2) increase incubation time with plasmid DNA and yeast cells keeping cells at 30C in the transformation medium may be overnight or more and/or also reduce the volume of transformation mix, increase the recovery time in the case of antibiotic selection. Also other suggestions in the thread are worth trying out.
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