1 reply to this topic

[SF_HK]

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

[T C]

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

  

SF_HK, on Apr 30 2009, 10:20 AM, said:

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