BAC recombineering - BAC elctroporation (Nov/21/2006 )
Hi fellow molecular biologist,
Here's a question/problem i have with BAC electroporation, if anyone can help i would greatly appreciate, so here's my problem:
When i electroporate my modified BAC in the pBR322 plasmid ( for subsequent ES cells targeting...) i always get mixed colonies, meaning that each colony has the pBR322 alone AND the pBR322-BAC, i don't know hoe to get ride of that damn pBR322 non modified!
Has anyone ever got the same problem?
Thanks for any kind of suggestions...
Cheers,
Criket.
you electroporate your DNA???????
may you reconsider your topic?
may you reconsider your topic?
Yes, i electroporate the pBR322 into bacteria that have been previously electroporated with BAC DNA.
I don't understand your remark, though.
Could you please clarify your question. And correct me if I am wrong
The problem is...
You are doing recombinarying between a BAC and pBR322.
Meaing.. you have first transformed your cells with BAC. Isolate cells which have picked up the BAC
Then later transformed those cells with pBR322
Activate the recombination machinery. (probably temperature rise to 37 Celsius)
Then isolate clones which have the recombined/fusion plasmid BAC-pBR322.. ie colonies which have taken up the pBR322
Only problem is all colony isolates contain both the unrecombined pBR322 and the recombined BAC-pBR322 plasmid.
If my understanding is correct, then the next step would be to re-transform the plasmid extract back into cells.
Ie you now have a plasmid solution which contain 2 types of plasmid.
You now put those plasmids mix back into bacteria cells.
As long as you don't use to much plasmid DNA, the bacteria cells will probably only pick up one plasmid, either ther pBR322 or the BAC-pBR322. You have to screen several colonies to isolate a colony containing only the BAC-pBR322.... as BAC-pBR322 is bigger then pBR322 so will transform less efficiently... fewer colonies will be BAC-pBR322 compared to pBR322
It would also help if you could tell me if the BAC marker is still working and as conformation is the pBR322 marker still working? If the BAC marker is different from the pBR322 marker, and is still functional, you can do a double selection on this tranformation step.
I had the same issue this week.
I diluted the mixed plasmid 1:1k and retransformed into DH5a and screened 20 colonies. Only 1 of 20 was positive for gap repaired plasmid ONLY.