Hi Vasantha,

my first question is have you used the right antibiotic?

As the prey plasmid ( I asume you have used pGADT7 or a devirative of this vector) has a ampicilline resistence sometimes people mix this up, however pGBKT7 has a kanamycine resistance.

If you have done this correctly another possibility could be that you kill your bacteria while inoculate your culture e.g. by using a inoculation loop which is to hot.

Another possibility is that you cells have lost the plasmid (some researcher report this, however I must admit I never had this problem), if this has happend there is not much you can do, except you have some plasmid DNA left of your contruct and you can retransform it.

You might also have killed all your cells while preparing your glystock or due to repeated freeze-and taw cycles. In this case I would use an amout of the glystock if you have enough ( I don't know how much you have) and try to isolate your plasmid directly as a plasmid miniprep and transform it in to competent cells.

I never have done this but you would only need a small amount of plasmid and I would think it is worth it to give it a try if everything else fails.

If you have a lot of dead cells You also have to make sure that you use enough of your glystock to grow the bacteria (but I don't think this is a major problem - usually a small amount is enough).

I hope this helps

Have you tried streaking out on non-selective media & selective media?

I typically use a sterile micropipette tip to streak - super cheap & no concern about accidentally cooking your bacteria with a flamed loop.

That will tell you if the bacteria are still alive (grows on plain LB) and if they still have the selectable marker (grows on Kan/amp/whatever you used).

Good luck!

What sort of cells did you transform your plasmid into? Most E coli should be very stable at -80, is the freezer working well? Or, any chance someone left your box outside the freezer? I had that happen to me once.