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PCR insert - in frame?

pcr in-frame insert

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#1 Koenholio

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Posted 29 July 2013 - 01:32 PM

Hi all,

 

I want to clone a PCR product with added restriction sites into a pLenti vector under an EF1a promoter. The forward primer contains the start codon (ATG) of the gene of interest, is this enough to get the insert in frame? If not, how do I make sure that my insert is in frame? Is this even important, since a frameshift would occur between the start and the stop codons, right?

Thanks in advance for your reply,

 

Koen

 



#2 Trof

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Posted 31 July 2013 - 03:10 AM

How is your specific vector designed, do you have a map? There are several types of pLenti vectors.

If your amplicon contains start and stop codons, you have the complete ORF in one piece, you can't be out of frame. You however need not to disrupt the promoter sequences upstream ORF, but these would be if you use recommended restriction sites

Some vectors may have fusion genes designed on either sides, which would require for example to omit stop codon and in that case you need to maintain frame at the end of the sequence (i.e. the reverse primer).

But no one can tell you unless you specify your vector.


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#3 Koenholio

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Posted 05 August 2013 - 12:29 AM

How is your specific vector designed, do you have a map? There are several types of pLenti vectors.

If your amplicon contains start and stop codons, you have the complete ORF in one piece, you can't be out of frame. You however need not to disrupt the promoter sequences upstream ORF, but these would be if you use recommended restriction sites

Some vectors may have fusion genes designed on either sides, which would require for example to omit stop codon and in that case you need to maintain frame at the end of the sequence (i.e. the reverse primer).

But no one can tell you unless you specify your vector.

 

Thank you for your reply.

That's what I thought, thanks :) the promoter is not disrupted - as you said, I'm using recommended restriction sites. Also, no fusion genes in the vector so that won't be a problem either.

 

Thanks for your help!







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