gDNA PCR - (Aug/19/2005 )
Is it necessary to shear or cut genomic DNA prior to PCR amplification of a specific site? or will denaturation @ 95C be sufficient?
If breaking it up is necessary, could anyone recommend a restriction enzyme to cut with if I want to amplify ~ 400 bp region? Would mechanical shearing with a thin gauge needle work as well?
-cwong1215-
You should not sheer your DNA because you will reduce the size of your potential targets and will not obtain longer amplicons. Use a good genomic DNA isolation protocol and check the 260/280 ratio of your DNA to make sure it is pure. Follow standard PCR protocols for the enzyme you are using. Good luck.
-tap14-
QUOTE (tap14 @ Aug 19 2005, 06:35 PM)
You should not sheer your DNA because you will reduce the size of your potential targets and will not obtain longer amplicons. Use a good genomic DNA isolation protocol and check the 260/280 ratio of your DNA to make sure it is pure. Follow standard PCR protocols for the enzyme you are using. Good luck.
yep. You should be able to pcr straight if you have good dna. don't use too much genomic template. If you get a smear of pcr product. You're using to much...
-vasussci-