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no bacterial growth after inoculation


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#1 astroid

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Posted 29 September 2011 - 05:10 PM

Hi everyone!

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!

Astrid

#2 allynspear

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Posted 30 September 2011 - 05:26 AM

This looks a lot like a very unstable plasmid, except that you are using StBL3 cells (good for unstable plasmids) and your insert is so short that your ligated plasmid is not that different from the vector itself (which I'm assuming doesn't have any trouble growing). I know this sounds strange, but you could trying picking a colony from your ligation plate and putting it on an LB plate without Amp. If the plasmid is unstable, you should still get growth, but your colonies will be plasmid free (or insert fee). If this is the case, I would try growing your transformants at 30 degrees and at 25 degrees, which slows down the bacterial growth and gives the bacteria more time to deal with an uncooperative plasmid.

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.

#3 pDNA

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Posted 30 September 2011 - 06:55 AM

how long did you incubate?
...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.

Regards,
p

#4 astroid

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Posted 03 October 2011 - 02:47 PM

Thanks for the answers!
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.

#5 allynspear

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Posted 04 October 2011 - 05:07 AM

StBL3 cells really shouldn't need 48 h to grow to a decent size, especially using Amp selection. I worry that you are trying to grow up satellite colonies that are an artifact of the high cell density that is plated in a typical transformation. I would transform some of your vector plasmid (uncut) into the StBL3 cells and see how long it takes for colonies to reach a decent size. Since your insert is so small, its unlikely that 57 bp would so dramatically affect the growth characteristics. If the vector transformants grow fine at 24 h, then your positive ligation transformants should grow within a similar time frame. I would say that you may need to revisit your ligation strategy and possibly your tranformation protocol.

Best of Luck.




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