Hi there,
as I'm really new in yeast, I got quite a basic problem with the two hybrid
system or better with my transformants. The amount of colonies I get I'm
quite content with, but I'm hardly able to reisolate any of the transformed
plasmids out of these positive colonies.
I use two strains: Y153 and HF7c (Clontech) together with the plasmids pAS2
and pGAD424. I transformed following the protocoll from clontech
(LiAc/ssDNA/PEG). (no record suspicious number of transformants, but
acceptable.)
To reisolate plasmids I tried two protocols: A.C.Ward (Nucl.AcidRes.Vol.18,
No.17, 5319) and R.Soni, J.Murray (Nucl.Acid Res. Vol.20, No21, 5852) as
well as a protocoll for direct PCR (G.M.Sathe et al. Nucl.Acid Res.Vol.19,
No.17, 4475) - all of them with really scanty or no results.
Both strains aren't leaky with LEU and TRP (the selection markers on the
two plasmids); I checked my media, retried with freshly prepared stuff...
It really should work!
Thanks a lot in advance for any advice!
Christine
froemel at cell.biol.ethz.ch
