I have tried to clone the 15kb plasmid (ligation product between 10kb and 5kb - cut gel extract). But after transformation into E.coli DH10beta and do the restriction analysis, we did not get the expected pattern. Even though, we reduce the temperature down to 30C which has been suggested to reduce this incident but the problem is still exist. Any idea that you guys could share?
Why do you think the problem is recombination and not merely that you never obtained the correct clone?
I cut both plasmids with two different enzymes, Kpn-I and Sal-I and we have purified the targeted fragments by excising from the agarose gel. After the ligation and transformation to E.coli DH10beta strain, miniprep and restriction digestion analysis, the patterns is not what we expected. As I discuss this situation with the colleague, he raised the issue on the rearrangement in E.coli since the plasmid is very large. So I am not quite sure whether there is any strain reducing the rearrangement, besides DH5alpha and DH10beta?
Both of those strains are deficient in normal homologous recombination so should be fine for most clonings. However some DNA fragments which have highly repetitive sequences can still be problematic and there are some strains that are further deficient in recombination, but these strains can be hard to manipulate.
However I suspect the problem is just that the plasmid is a bit large and you are mostly going to be selecting for smaller things. I have had similar problems in the past and found that if I just screened a larger number of colonies after the transformation I often found what I was looking for. So before worrying about a different strain too much, I would just repeat your cloning experiment and screen more colonies. Also it may be that the right clone makes the cells grow a bit slower, so after transformation be sure not to just pick the largest colonies on the plate, pick a variety of different colony morphologies and you might find that the slow growing smaller colonies were the correct ones, if you had variation.
Thank you so much for your comment. We got >100 colonies, we screened 30 colonies and we did not get the positive yet. May I also ask further question what frequency you will get the positive clone with the large plasmid?
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