gene instability or gene product toxicity?

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nesma7
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gene instability or gene product toxicity?

Post by nesma7 » Wed Jul 23, 2008 12:35 am

I synthesized a gene of 192 bp coding for miniproinsulin by a gene synthesis company.
the gene was sent to me in pCR4-TOPO vector
cloning that vector in DH5α or Top 10 E.coli was not sucessful {didn't give the expected size of the plasmid & did not contain my restriction sites ( BamHI & HindIII )}.

I decided to change the vector
- by cloning the gene in pQE-30 Xa ( Qiagen ) ,the ligation was successful -
transforming the ligation product into DH5α was not successful .also to M15 & Top 10 .(no colonies appear in the plate) -( regarding that +ve & -ve control plates are good ).


Do you think it is some kind of insert instability due to miss translation from amino acid to nucleotide during the gene synthesis? Or it is a gene product toxicity ?
& what can I do ?
Could any one help ....?

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