Ecoli genomic recombination causing cloning problems - after ligation vector size and sequence change (Sep/20/2006 )
I have the following problem. I want to subclone a 1.7 kb cDNA into a 15 kb eukariotic expression vector with a 8.5 eukariotic genomic promoter sequence (sticky ends). After ligation and transformation I get a reasonable number of colonies (10-100) but no colonies in the negative control. However, when I get my minipreps the plasmid runs at a much smaller size than it should and it is very hard to cut it with restriction enzymes. By PCR and sequencing I did confirm though that my cDNA is in the plasmid, but when I sequence out of my cDNA into the vector I get Ecoli genomic DNA instead of eukariotic promoter sequence. I assume that some sort of genomic recombination took place in the Ecolis, but I never experienced this problem before. I used XL-10 gold (Stratagene) and Stbl2 (Invitrogen) Ecoli strains. My next step is to use SURE Ecoli from Stratagene, but I am not so optimistic this will solve the problem.
If anyone of you experienced a similar problem and has any suggestions as to how to address the problem, your help is greatly appreciated.
I have experience similar a problem, though with larger vectors... BACs
Assuming this is a recombination problem, I have several suggestion, some of which might be helpful.
Most importantly, clone your sequence into a single copy plasmid. A small BAC would do fine, like BACe3.6.
Grow your plasmids in a strain like DH10B. Those things keep large, unstable vectors rather well.
Grow your cells at reduced temperature like 34 Celcius and grow large volume of cell to make up for the reduced conditions.
Don't try to keep the plasmid in cells, even recombination deficient, low copy number plasmids will recombine given time.
Hope this helps
SURE cells (stratagene) should help. Stbl3 or DB3.1 from invitrogen may help.
Grow ur cultures for not more than 12-14 hrs. Longer times will facilitate recombination events.
Thanks so much people!
So far I transformed SURE Ecoli and I will try low temperature/large volume/short incubation LB cultures for my minipreps. I will keep you updated about the results. I also did some more research on the web and I found that some people address the problem of genomic stability vs yield of plasmid by constructing low copy plasmids that can be induced to high copy number. The most interesting one I found is called pSCANS (http://www.genome.bnl.gov/Vectors/index.php), but there also is a fosmid pCC-FOS. Finally there appears to exist an Ecoli strain that can basically do the same, i.e. it will propagate high copy plasmids at low copy number but canthen be induced to high copy number plasmid expression (http://www.epibio.com/pdfforum/11_5_6-7.pdf#search=%22inducible%20copy%20number%20plasmid%22). Sounds good to me, actually almost too good to be true... Does anyone have any experience with these products?
So here is an update.
It seems the SURE Ecoli from Stratagene did work better, roughly 50% of the minipreps (growing at 30C for 15 h) show the right size plasmid and I can cut out my inserted cDNA. Sequencing data are still missing, but so far it looks quite promising. However, I find it still amazing that even in these Ecoli I see quite a high percentage of recombination with my 17 kb plasmid, which is after all not that big. The copycutter Ecoli did not perform better, i.e. they too have recombination mixed with normal plasmid, but their transformation efficiency appears to be lower than that of the SURE Ecoli. They grow at a much slower rate than SURE cells, so I guess that the principle of keeping even pBluescript vectors at a low copy number seems to work, after induction to high copy number for 4 h the plasmid yield still is ~5 fold lower than with SURE Ecoli.
So far, thanks again everybody for your advice,
I got the correct sequencing result. Sure2 E.coli works pretty well though I got some recombination (~70-80%) mixed with normal plasmid. As long as screening more colonies in the miniprep, I could get the correct clone. Thanks guys for helping.