Replacing of GFP cloned inside a gene with RFP - (Aug/01/2006 )
Hi All 
I got one working binary vector from some other lab. There is one gene cloned and GFP gene cloned inside this gene. Now we want to replace this GFP with RFP. GFP is cloned in NcoI and Bam HI, and the RFP which I am going to clone got EcoRI (just before start codon) and Bam HI (just after stop codon)ends. I think I have to mutate this stop codon before cloning since I am cloning into another gene.
If so please someone would suggest how to do mutation and what would be the best kit.
One brief description of whole cloning strategy would be a great help for me.
Thank you
Hi,
You can design a primer with the Nco I changed to EcoRI for the forward. As for reverse, don't have to change anything, just design primers at the 3' end inclusive of the Bam HI sequence. Try designing so that Tm is above 65 to 70C, it'll cause less problems. PCR the construct that you have, gel purify the correct fragment, digest with the REs, gel purify again, then ligate to the vector you desired.
Oh ya, these too:
1. Make sure the 3' end of primers are NOT G or C.
2. Add two nucleotides, eg. GT in this sequence just before the RE site; GTGAATTCyour sequence.
3. Primer dimers are minimal (use IDT to determine)
etc...
Cheers
You can design a primer with the Nco I changed to EcoRI for the forward. As for reverse, don't have to change anything, just design primers at the 3' end inclusive of the Bam HI sequence. Try designing so that Tm is above 65 to 70C, it'll cause less problems. PCR the construct that you have, gel purify the correct fragment, digest with the REs, gel purify again, then ligate to the vector you desired.
Oh ya, these too:
1. Make sure the 3' end of primers are NOT G or C.
2. Add two nucleotides, eg. GT in this sequence just before the RE site; GTGAATTCyour sequence.
3. Primer dimers are minimal (use IDT to determine)
etc...
Cheers
Thank you very much for your replay. Let me ask few more qustions i fyou dont mind.
I hope you mean I should design a primer Eco RI changed to Nco1 because when I digest out GFP from my vector, I will be getting NcoI and Bam HI site on my bector and my RFP fragment got EcoRI and Bam HI site.
Any way I got a new problem now. My RFP gene got one NcoI site inside the gene. What I am planning now is I will select another enzyme which produces NcoI overhangs and I will design a primer accordingly. Please comment on my idea.
Is it necessory to clone my RFP PCR fragment to another plasmid before clone to my vector because pBIN binary vector is very large.
Is there any need to dephosphorylate my vector before cloning my PCR fragment?
THANK YOU