transformation competent cells problem - fastbac vectors (Jul/12/2007 )
hi
i'm new to Fastbac vectors. I was given a Fastbac cloned construct. I was new to carry transformation in DH10 Bac cells (competent cells harbouring bacmid). The plasmid DNA (fastbac constuct)was kept at 4 deg fridge for a month. I tried doing transformation with heat shock. Transformed the cells to a antibiotic plate and tried doing IPTG/X gal selection for the recombinant plasmid. I did not see any colonies, neither blue or white. I have doentransformation with E.coli competent cells. It worked for me.
This new system dosen't seem to give results. Could the DNA be denatured if it is kept at 4 deg for a month. Is there a way to trouble shoot this?
-mini-
QUOTE (mini @ Jul 12 2007, 04:01 AM)
hi
i'm new to Fastbac vectors. I was given a Fastbac cloned construct. I was new to carry transformation in DH10 Bac cells (competent cells harbouring bacmid). The plasmid DNA (fastbac constuct)was kept at 4 deg fridge for a month. I tried doing transformation with heat shock. Transformed the cells to a antibiotic plate and tried doing IPTG/X gal selection for the recombinant plasmid. I did not see any colonies, neither blue or white. I have doentransformation with E.coli competent cells. It worked for me.
This new system dosen't seem to give results. Could the DNA be denatured if it is kept at 4 deg for a month. Is there a way to trouble shoot this?
i'm new to Fastbac vectors. I was given a Fastbac cloned construct. I was new to carry transformation in DH10 Bac cells (competent cells harbouring bacmid). The plasmid DNA (fastbac constuct)was kept at 4 deg fridge for a month. I tried doing transformation with heat shock. Transformed the cells to a antibiotic plate and tried doing IPTG/X gal selection for the recombinant plasmid. I did not see any colonies, neither blue or white. I have doentransformation with E.coli competent cells. It worked for me.
This new system dosen't seem to give results. Could the DNA be denatured if it is kept at 4 deg for a month. Is there a way to trouble shoot this?
We have our plasmids in 4C for months even years and as long as they are not contaminated, they should be fine. Run the plasmid on the gel. You would see if its degraded or not.
-scolix-
yep, idea looks good. will try out.
-mini-