Recombinant plasmid lost afer transformation - (Aug/28/2007 )
hi.... now i'm working in yeast project.. i've got my ligation product in pYES2/CT (invitrogen) vector. it is a shuttle vector.. before i insert it to yeast cell ; S. cerevisiae INVSc (Invitrogen), i checked all samples by running on agarose gel and cutting them with restriction enzyme (EcoR 1), the result shows they were in a good condition. I transformed it with LiAc/PEG/TE procedure (Invitrogen), i used 10 ul of samples.. after 4 days incubation in 30 degrees, some colonies grew on plate medium. As procedure i got, we must transform the extract plasmid from yeast to E. coli. So, i extracted the plasmid from all transformant cells then i transformed it to E. coli, i used 10-20ul samples/tube e.coli competent. After 16 hours incubation, i saw E. coli colonies on plate (SOB medium with ampicillin 100mg/ml). I checked E. coli transformant by extracting plasmid, running on agarose gel, cutting them with same restriction enzyme (EcoR1).. I've done for several times... the problem is.... in my first experiment i could get the plasmid, but i can't get it anymore... is there something wrong with my experiment?? i'm so desperate... i need replies as soon as possible... thanks in advance
I think the key is likely the transformation of the plasmid back into E. coli. You said you were adding 10-20 ul samples to your tranformation cells. This is 10-20x too much. Try it again adding 1 ul of your plasmid to 50 ul of cells, SOC, growing up for 1 hour at 37 with shaking, and plating out on selective plates.
Less is more.
Less is more.
thanks for ur suggestion. actually dna samples that i used to be inserted to E. coli is plasmid recombinant from yeast transformant. when i added 1 ul to E. coli, it doesnt grew on plate. may be the amount of yeast plasmid is lower than ussual. w
in the other hand, after i added 10-20 ul of yeast plasmid recombinant to E. coli. E.coli was grew up. unfortunately i couldnt see any band of my plasmid extract from transformed E. coli on agarose gel. i used plasmid without insert as positive control for yeast and E. coli transformation. i saw band on my positive control after i extracted it and visualized it on agarose gel, but other no. negative control of my transformation didnt grew, means my tansformation was doing well.
wat should i do?? or my plasmid recombinant from yeast doesnt stable enough in E. coli???
The retransformation back into E.coli could be tricky. There could be several possible problem, though:
- You said that the original construct is OK (the one you used to tranform into yeast). Are you sure that that construct is succesfully transformed into yeast? As in, how did you check the yeast colonies that you got after transformation? Did you check by protein expression (did the construct express in yeast)?
- Assuming that the construct is expressed alright in yeast, after you extract them back out from yeast, what method did you use for this step? Is the extracted plasmid good? Is it the same as the original one (as in, did you check it by digestion or so)? We usually use phenol for this extraction, so is the quality of the extracted plasmid good? Did you run gel and check it?
- When you retransformed back into e.coli (although I wonder why?), how did you do transformation? To our experience, electroporation is the much recommended method.