a couple of questions about cloning.

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oppox
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a couple of questions about cloning.

Post by oppox » Fri May 26, 2006 10:48 am

Im having troubles with a gene, an AT rich gene.
problem 1.
I have cloned my gene (700 bp) into a petblue vector (novagen), an open vector with a T overhang making it possible to ligate it with any fragments amplified with taq poly. I have the correct insert according to sequencing, but I cant express the protein. The vector demands special cells for expression because it lacks the lacI repressor. The gene contains alot of rare codons but even with the rosetta cells not a single tiny protein is produced. Does anyone had the same experience?

Problem 2.
Same gene different vector (pET28b).
Here the restriction digestion doesnt work, cutting the gene and vector with BamH1 and NDE1 (BSA as buffer). No insert in any of the clones I get (Kanamycin resistance). Anyone?
thx in advance

tranjo
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Post by tranjo » Sat Jun 03, 2006 4:13 pm

Try BL21(DE3) for expressing proteins. I don't know the genotype of that strain, but I've used it top express maltose-fusions before. Also, your protein maybe lethal to the bacteria when expressed.

Both enzymes cut fine in NEB2 supplemented with BSA. BSA does not inhibit reactions. I hope you made a typo when you said "BSA as buffer." BSA as a buffer would give you nothing.

Best, J

oppox
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Post by oppox » Wed Jun 07, 2006 7:40 am

oh yea, it was a typo, I meant BSA is added to the buffer :)
Well as of now I think we are leaving the conventional cloning procedure and try out the ligase free cloning instead. Thx for the help.

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