Vector with many restriction site - (Jan/23/2007 )
I got one vector with GFP gene cloned in NcoI -BamHI site. I would like to replace the GFP with RFP in the same site. The problem is the vector consists 3 more Nco1 site as well as 2 more BamHI site. If I go for partial digestion is it possible to get backbone which is free of GFP gene (digested out) for cloning for RFP? Please post your comments. Thanks in advance........
I don't think you should attempt the partial digest. this digestion stretagy is too complicated for a partial digest. Do not attempt
How big is your vector? You could PCR amplify the vector backbone. thus obtaining the backbone this way.
Any idea how the GFP got into the vector in the first place? The GFP must have been added rather early in the vector's construction.
I would PCR amplify RFP and clone it into ur vector with the other adjacent sites.
Hi perneseblue, I already contacted u for this same cloning. This cloning looks very simple and practically very complicated.I am keep on trying different strategies nothing works.
GFP is cloned in a hydrophilic loop of another gebe and these 2 genes are cloned in pBINPLUS on Sal1 site. So excluding GFP, the back bone is around 18kb. we got pfu in our lab. I heard from someone that pfu mixed with taq may push to ampilify such a big backbone. U have any idea? Whats your preferential polymerase to amplify such a huge backbone?? Please help me ....
How big is your vector? You could PCR amplify the vector backbone. thus obtaining the backbone this way.
Any idea how the GFP got into the vector in the first place? The GFP must have been added rather early in the vector's construction.
but scolix, the problem is GFP is cloned in another gene and we suppose to ecpress both the genes together. If I select some other site, I am afraid it may change the reading frame 
Isn't it possible to cut out your GFP region with other restriction sites, clone it into pUC or pBlueskript (or any other cloning vector), then either by PCR or restriction remove the GFP and replace with RFP, then clone the entire region back into your larger vector? I've done comparable stuff with 15 kb vectors (bear in mind that GFP is only 800 bp or so, how on earth are you going to make sure you have removed single cut vector before ligation? They won't separate on agarose gels...? I used about 4-5 kb into a cloning vector and it was hard enough to remove single cut vector as it was (but you only need one correct clone, so having high background isn't too much of a problem).
In this case, I would agree with vairus's suggestion. Clone the fusion construct into a cloning vector and then exchnage the GFP for RFP and then clone the whole fusion construct into your original vector.