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plasmid incompatibilities with certain sequences


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#1 sparky

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Posted 30 August 2010 - 12:24 PM

Hi,

I am trying to clone a triplet repeat sequence (30 - 100 rpts, total) into a pUC-based vector. I have tried both TOP10 and XL2-Blue cells, but both have kicked out most or all the repeat region. Can anyone give me a better genetic background in which to try cloning or is there perhaps such a thing as a vector issue? By vector issue, I mean do I need to get perhaps a new inducible promoter or different antibiotic selection gene? The actual promoter on the clone that would be linked to the repeats has not been activated. The only gene so far selected has been amp.

Any advice would be greatly appreciated.

Sparky

#2 phage434

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Posted 30 August 2010 - 03:43 PM

You should first clone into a low copy number vector, such as a pSC101 origin or F plasmid origin. You should use a repeat friendly strain, such as SURE or STBL2. You should grow at lower temperature, such as 25 or 30. You should (as you are) avoid inducing expression. Those are about all the tricks I know.




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