Hi all, I am struggling to transform my BY4742/1 cells using the Gietz protocol along with a linear PCR fragment. I understand that using a PCR gene disruption cassette is not as efficient as using a plasmid for homologous recombination however this is the situation I find myself in. My fragment is ~ 2kb and I have used a high fidelity pol for amplification. I'm wondering if anyone has any experience in doing a gene deletion this way and how much DNA & ssDNA carrier you used for a successful transformation. Would it possibly be better trying electroporation and if so how much DNA would be suitable in that situation?
Thanks!!
Dear Jessica Reid
I perform transformations in BY4741/BY4742 yeast cells using both linear PCR cassettes and linear integrative plasmids. The advantage of using linear plasmid DNA is its transformation efficiency is higher and less DNA is required for transformation (600-1000 ng per transformation reaction). Transformation with PCR cassette fragment requires higher concentration of DNA in the transformation mixture. It is recommended to use 1-2 μg but sometimes it works even with higher amount of DNA (3-4 μg), specially when primes contain strong secondary structures.
I am writing down here a protocol which we use in our lab and the transformation efficiency is very well.
- PCR fragment DNA/linear plasmid - X μl
- Carrier ssDNA (2 mg/mL) - 50 μl
- 1 M LiAc - 36 μl
- Distilled sterile water - (34–X) μl
- 50% Polyethylene glycol - 240 μl
- Total volume - 360 μl
We use Salmon sperm DNA as carrier ssDNA. I wish you all the best.
Best regards
Satyendra
Satyendra Mondal A quick technical question if you don't mind - Do you use purified PCR fragments from an agarose gel or do you purify straight from your PCR products?
Thanks again for the help and much needed insight!
Kind regards
Jessica
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