ligating a 6 His tag to plasmid - (Jul/06/2011 )
hi evryone
I want to insert a 6 His tag into EGAD CFP plasmid. The tag needs to be in front of the CFP. there is a AgeI site in front of the CFP.
my proposal: cut the plasmid with AgeI, fill in the overhangs, A-tail the ends, T-tail the 6 His tag and ligate. now my question.. can this be done????
if yes what problems will i face and is there a better, safer and easier way???
in between: my original EGAD plasmid is with GFP. i am planning to swap the GFP in the plasmid with CFP from a clonetech plasmid.
thanks in advance!
-sou-
sou on Wed Jul 6 14:28:53 2011 said:
hi evryone
I want to insert a 6 His tag into EGAD CFP plasmid. The tag needs to be in front of the CFP. there is a AgeI site in front of the CFP.
my proposal: cut the plasmid with AgeI, fill in the overhangs, A-tail the ends, T-tail the 6 His tag and ligate. now my question.. can this be done????
if yes what problems will i face and is there a better, safer and easier way???
in between: my original EGAD plasmid is with GFP. i am planning to swap the GFP in the plasmid with CFP from a clonetech plasmid.
thanks in advance!
Hi there.
Hopefully this isn't too late of a reply, but have you considered using a QuickChange-type approach, whereby the 18 extra nucleotides are introduced via a megaprimer?
-omp-
hi thx for the reply... and that is exactly what i did.. ordered the primers last week..
fingers crossed! 
-sou-