transformation of Saccharomyces cerevisiae - (Nov/04/2008 )
Plz suggest the amount of vector and PCR product to be used for transformation in S. cerevisiae. I am a bit confused with the variations in the protocols available on net.
some suggest the total dna should be 1ug - 5 ug in 1-5ul of volume, but this could not be possible in my case, as i dont have E coli clone, but i am going directly with my approach:
1) Vector + gene digestion with same RE
2)vector + Gene Ligation
3) linearization of ligated product (Partial digestion)
4) transformation with SC easy comp transformation kit (Invitrogen)
But the problem is finally i am getting a 0.18ug Dna (vector + gene) that too in 18 ul of volume which is against recommended (1ug - 5 ug in 1-5ul of volume) in the kit.
waiting for ur valuable suggestions
I have used homologous recombination with the S.c easy comp transformation kit. I added 1 ug of gapped vector (RE double digested) and 5 ug of insert DNA per 100 ul of competent cells. The key is to have identical 50bp overhangs at the ends of vector and insert. The way I did it is to add 50bp overhangs to either ends of the insert PCR product. The PCR would be difficult in this way but you can seperate the total about 70bp into two pieces: 50bp overhang and 20bp overlapping region + the actual primer. Good luck!
Your approach is extremely hard because:
1. the total amount of ligated product is too little. Transformation of even bacteria is really difficult with the ligation product. Not to mention you have an extra partial digestion step, and yeast is more difficult to transform than bacteria.
2. The SC kit does not have high transformation efficiency even with intact plasmid. The efficiency with linear DNA is higher but I think it is still way less than sufficient.