in frame problem after 3 months of cloning! - can I stil use my construct (May/21/2009 )
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
SF_HK on May 21 2009, 04:23 PM said:
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
I believe that if you dont have a stop codon your protein will be "tagged" with whatever the sequence becomes with this new T until the next stop codon.
I know is not good news, but I'll re-clone it.
I might be wrong thou, so more advice here will be welcome.
I agree. Presumably your second try at cloning will be a lot quicker now that you know how to do it.
You can also try something like Quick Change mutagenesis kit from Stratagene.
I tried the mutagenesis kit form Strtagene. There were 20 colonies picked two and the extra nuleotide wasn't deleted. Will pick all the 20. I have a feeling doing a deletion is more complicated than a mutation. I did a single base pair mutation last week and got 100% efficiency. All my clones had the mutation.
Why did it take two months to clone your insert?