subcloning problem - inframe fusion (Feb/04/2010 )
I have cloned a gene into PTZ57R/T vector by INSTA TA cloning kit.
now, I want to subclone it into PRT100 vector (plant expression vector). So, I will digest both the (PTZ57R/T + gene) and PRT 100 vector with XbaI and Bam HI restriction enzymes which will release the gene insert from PTZ vector so that it can be cloned at XbaI and Bam HI site of PRT 100 vector. My doubts are
1.I think inframe fusion is very imp as i am cloning it for expression purpose.
but ,becoz i have initially cloned into TA vector which will definately have Tand A overhangs ( which will be included after digestion) may disrupt the inframe fusion . So, should I consider this overhang for inframe fusion or not.
2.Should the PRT 100 vector be given CIAP(CALF INTESTINE ALKALKINE TREATMENT OR NOT).
Thanx a lot
As far as I remember correctly the poly A tail comes after the stop codon. So it shouldn't matter if it gets included in the expression plasmid. Maybe someone else can verify this.
I wouldn't treat the cut vector with CIP. The vector ends after ligation aren't compatible. Usually vector ends are treated with CIP to reduce self-ligation. The generated ends are cohesive and not compatible.