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inframe problem


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#1 SF_HK

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Posted 29 April 2009 - 08:50 PM

Hi,

It took 2 months to clone my insert in pcdna3.1(-)/myc/his (invirogen) vector. However, upon sequening I realise a problem.

I wanted to tag my full length insert and thus did not include the stop codon. Upon sequencing I find that he forward sequence is in frame but I have an additional nucleotide at the end of my sequence? so:

cdna sequence: 5;attTAG (TAG= stop codon)

my intended insert seg: 5'-att only followed by the vector sequence

my wrong insert seq: 5'-attT-followed by the vector sequence (so basically, I know have the additional T from the TAG that I intended to delete)

So, I obviously won't be geting my tag but can I detect my protein using the protein antibody? Or wil my protein be affected?

Thanks

#2 T C

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Posted 30 April 2009 - 12:30 AM

Hey,

I think it should be okay. Your protein would be translated in the right frame but would have a C-terminus that would come from the vector but in a different frame.

Hope it helps.

Best,
TC



Hi,

It took 2 months to clone my insert in pcdna3.1(-)/myc/his (invirogen) vector. However, upon sequening I realise a problem.

I wanted to tag my full length insert and thus did not include the stop codon. Upon sequencing I find that he forward sequence is in frame but I have an additional nucleotide at the end of my sequence? so:

cdna sequence: 5;attTAG (TAG= stop codon)

my intended insert seg: 5'-att only followed by the vector sequence

my wrong insert seq: 5'-attT-followed by the vector sequence (so basically, I know have the additional T from the TAG that I intended to delete)

So, I obviously won't be geting my tag but can I detect my protein using the protein antibody? Or wil my protein be affected?

Thanks






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