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cloning in pQE30


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#1 shilpamadhu

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Posted 21 January 2005 - 06:21 AM

hi,
I am not able to clone my pcr product in pQE 30 for expression. On the gel and in transformation results, it looks like digestion and dephosphorylation is working.
When I do the colony PCR for the presence of insert, I get right sized fragment. But when I do PCR for vector specific and insert specific primers, I get wrong bands. I also got my clones sequenced, but result shows no inset sequences.
could someone please help me ???

Thanks
Shilpa

#2 mybbff

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Posted 21 January 2005 - 07:30 AM

The template you used in colony PCR may be contaminated by the vector which has no insert in it, or by the insert fragment to be cloned into the vector. Namely false positive results. Then you'd better identify your recombinant clones by plasmid-extracting, followed by PCR or restriction with proper enzymes.

Good luck!

#3 suhasbn

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Posted 21 January 2005 - 08:29 AM

it might be just another case of false positives..........! it happens to all of us. just try to pick many more colonies that too far and isolated ones. one of them shud have ur insert.
gud luk

#4 shilpamadhu

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Posted 24 January 2005 - 06:51 AM

thanks very much!!!!!




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