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Cloning: dealing with a toxic sequence?
Posted by: archteryx (IP Hidden, New member, 0)
Date: June 25, 2002 06:50PM
I've run into a problem that I don't have enough experience to crack -- a toxic sequence during a large cloning project.
The toxic sequence results when two pieces are combined: a small (400 bp) and large (4.5 kb) fragment. Both fragments will clone fine by themselves. Combined, they form a toxic sequence and appear to be unclonable. So far, I've tried: -- Different hosts strains of E. Coli (SURE, DH5alpha). -- Different vectors for both pieces, by subcloning, then combining in different vectors. (tried pUC19, NEB Litmus 29) -- Different surrounding sequences of both pieces. -- Doing the transformation and subsequent grow-out at 30 degrees instead of 37 degrees, as suggested in an earlier post. Unfortunately, I cannot simply toss the small piece out...it is required in my project. Nor can I make large deletions or other *major* alterations to its sequence. What other techniques are there for cloning these sorts of "toxic sequences" besides lowering the temperature of incubation? -- Cody Buchmann
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