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difficult cloning of large insert - (Apr/05/2007 )

I am trying to clone a largeish gene (~15 kb). I have PCRd it up in 4 parts and cloned each quarter into a seperate cloning vector. So far I have managed to paste 3/4 of it together using internal restriction sites, although the E coli containing the plasmid with the 3/4 gene grow very slowly and the plasmid copy number seems to have dramatically reduced. I am finding it impossible to clone the last part of the gene in. My transformed cells keep reverting to empty vector. There is nothing on the cloning vector to suggest that the protein can be expressing and making the cells sick therefore is this just a size issue? Do the commercially available bugs that claim to support large plasmids work? Does anyone have any suggestions that might help me?


For the cloning of large constructs, we use XL10gold from Stratagene. It works very well for constructs of 21 kb that we have now. According to Stratagene, it can hold up to 34 kb, but we haven't gone so far yet. The biggest we have cloned into the cells was 21 kb. We then also have low recombination numbers.


I grow my vectors that are about 15-16 kb in DH5alpha cells quite happily. They do contain viral LTR-sequences (very unstable) which is why we grow them on 30°C instead of 37°C and at milder shaking. Yields decrease, but are acceptable, if you grow them longer.



Could you guys please help me..!

Though the insert I am trying to clone in is not as large as yours(its just 2.2 Kb), the high AT content really cause me a lot of problem while cloning.
I use invitrogen gateway cloning , and after the first recombination reaction I get a lot of colonies on antibiotic plates, but none of them are positive..!
I am afraid, may be DH5alpha I use for cloning must ve chewed my plasmid up becoz of its high AT content....could you please tell me, which strain I shud use to overcome this problem?