How to remove a few bases from my cloned insert using PCR - (Apr/20/2007 )
I have a clone in a vector. I want to remove a few bases to bring it in-frame. How will I remove these bases using PCR?
there are many ways to skin a cat, thus similarly there are many ways to solve your problem, most depend on the exact nature of your vector and the location of said basepairs in relation to a unique restriction site?
If your vector is small ( I understand more experienced members would gladly do this protocol on any plasmid smaller 10kb) you can try removing said basepairs bysite-directed Mutagenesis, which uses PCR, primers that misses out the offending bp and DpnI treatment to cut the old plasmid.
Another way of fixing said error (though in a less elegent manner),
If a unique restriction site is close by (less then 100bp) (This site is called A)
It is simple matter of building a primer without the offending bp, but contains that unique restriction site. PCR amplifiy between the region between the unique site near the offending bps (A) to another unique restriction site further along (Called B). Since the primer does not have the offending bps, the PCR product is would be inframe. Cut the vector, at (A) and (B), and paste in the PCR product. The plasmid should then be inframe.
Another way of doing this
Would be to reassemble your gene from scratch using PCR. Lets say your bp are in the middle of the gene. You build 4 primers. Primers (1) and (2) go right up to the offending bp. With primer (2) missing the offending bp. Primer 2 also has a little overlap shared with primer (3). Primer (3) and (4) amplify the remained of the gene.
You then fuse the PCR products of (1) and (2) with (3) and (4) by PCR. Product fusion is possible because of the overlap shared between primer (2) and (3).
If they are in the middle of the
Thanks Pernese Blue. I have never performed Fusion PCR. I tried looking for an illustration over the net. Do you know of some good site that may give an illustrated example.
Unfortunately I don't know any sites.
But the above topic contains a discussion on "fusion by PCR" I had. I drew a nice illustration too.
If you have questions, don't hesitate to ask.