no bacterial growth after inoculation
Posted 29 September 2011 - 05:10 PM
My cloning strategy involved the annealing of oligos (57 bp) and the ligation with the prepared vector backbone (8.7 kb).
After the ligation and the transformation into Stbl3 cells I got more than 20 colonies on my 2 sample plates and not even one on the religation control. The problem is now that after the inoculation of 4 colonies of each plate I didn't get any bacterial growth. Not on the backup plate and not in my LB medium (containing Amp), which was gonig to be used for minipreps.
So far I have done two transformations, the second one again showed some colonies on the ligation plates and nothing on the religation. Again I picked 4 colonies each for the inoculation and didn't get any bacterial growth.
The last inoculation lead to the bacterial growth in one of the flasks (and on the associated part of the backup plate) but nothing in all the others.
I have used the same batch of LB + Amp plates for all my incubations and also the same LB media and concentration of Ampicillin for the flasks.
Nothing like this has ever happened to me and I can't think of anything that could have went wrong.
Any suggestions are highly appreciated!
Thanks a lot!
Posted 30 September 2011 - 05:26 AM
It is also possible that your competent cells are not playing nice. I still have no explanation, but we had an entire batch of competent cells that would transform well, but then lose the plasmid almost every time we tried to re-streak or re-grow cultures. We ended up throwing the whole batch away and making new competent cells, which solved the problem. I wish I could explain it, but you might just want to try a different batch or strain of competent cells (borrow from a neighboring lab if you don't have others).
Best of Luck.
Posted 30 September 2011 - 06:55 AM
...i saw clones that needed more than 24h to show any turbidity ...so i would try to incubate longer if you haven't done so yet.
Maybe they just have a very low growth rate or suffer from plasmid instability.
Posted 03 October 2011 - 02:47 PM
I have already tried to incubate it even longer than 72 h as the colonies on the ligation plates also needed 48 h to grow to a proper size.
I will try to grow it on a plate without Amp and also try a new batch of Stbl3 cells and see what happens.
Posted 04 October 2011 - 05:07 AM
Best of Luck.