If you are growing your culture at 42 and then speaking out on plates with and without Gent, in order to have no growth on the Gent plate you need 100% curing. This might not be occurring.

I would grow for the 48hrs at 42C and then streak out or plate for single colonies on plates without Gent. Then test 10 or 20 colonies from those plates to see if they are still Gent resistant. That way even if only half the cells are cured, you are still likely to find one or two cured colonies (and it should be much higher than that).

Michael J. Benedik Thank you for the answer!

Actually, I've already tried 'individual colony picking' from streaked plates but all the individuals(colonies) still have Gent.. they keep growing.

Is there any possibility, whether Gent somehow integrated to other part of genome such as DH10B g-DNA or Bacmid genome, so it can not get Gent off.. ?

This is so confusing,,

Thank you for reading :)

It's hard to know what happened without a lot of analysis. But let me throw out some things to confirm:

1) when you grow out your culture at 42C I presume you do not have Gent in the media?

2) Are you sure the gent plates are working (ie the gent is still good)

3) you might just try and repeat the whole thing again, on occasion something weird happens but doesn't repeat

Michael J. Benedik Thank you! These are the answers.

1) yep! I used 'Kan+Chloram' LB medium for pKD46 curing (did not use Gent)

2) All plates i used have no problem, i checked with another test plasmids

3) yes.. since it's about 'Biology' anything weird can happen. i will just try all over again.

Thank you so much!! :)

Good luck

Try at 37? :D