Help for my cloning - xba 1 and not 1 digetion (Apr/09/2008 )
hello everybody,
i am really gettign a great problem!
i have a gene which i have to subclone in to pCDNA3 vector at Xba1 and Not1 sites (i have no other choice). these sites are not present in the original construct. so i designed forward and reverse primers with Xba1 and Not1 sites respectively. did PCR and nice band came out. I digested it with NEB3 buffer with Not1 and Xba1 together for 2 hours. the same procedure was done for pCDNA3 vector. after the double digestion, i dephosphorylated my vector with Shrimp AP for 20 minutes. digested PCr product and dephosphorylated vector was gel purified and I ligated both of them in 3:1 ratio using T4 DNA ligase (NEB), for 20 minutes (as by NEB suggestions) and finally transformed e.coli. next day i got nearly 100 colonies but my problem is i m not getting the positive clone after mini restriction digestion analysis. I have tried 2 times this procedure and failed. can any bosy help me plz??
Did you do a vector only control?? and if yes, did you get any colonies??
i am really gettign a great problem!
i have a gene which i have to subclone in to pCDNA3 vector at Xba1 and Not1 sites (i have no other choice). these sites are not present in the original construct. so i designed forward and reverse primers with Xba1 and Not1 sites respectively. did PCR and nice band came out. I digested it with NEB3 buffer with Not1 and Xba1 together for 2 hours. the same procedure was done for pCDNA3 vector. after the double digestion, i dephosphorylated my vector with Shrimp AP for 20 minutes. digested PCr product and dephosphorylated vector was gel purified and I ligated both of them in 3:1 ratio using T4 DNA ligase (NEB), for 20 minutes (as by NEB suggestions) and finally transformed e.coli. next day i got nearly 100 colonies but my problem is i m not getting the positive clone after mini restriction digestion analysis. I have tried 2 times this procedure and failed. can any bosy help me plz??
Regarding the design of your primers, did you add some extra bases at the end to allow the restriction enzymes to efficiently cleave your PCR products?
Also, did you gel purify your digested vector DNA to make sure there was no supercolied (i.e. uncut) vector carried over into your ligation? I'm amazed at the number of people who don't do this. You only need a trace amount of undigested vector to give you huge numbers of empty clones after transformation.
Ginger
You can, of course, completely avoid the plasmid carryover by making the vector backbone by PCR instead of by cutting circular plasmid. This eliminates a lot (essentially all) of the background.
I've tried this before and often have problems getting product from the PCR reaction. I imagine this is due to the size of the vector (typically 4-8 kb). Do you have any suggestions on how to get good PCR results? Thanks.
Phusion master mix. Follow their recommendations for cycle conditions.
Note: XbaI is blocked by overlapping dam methylation. To remove dam methylation use a dam deficient strain.