Enzymatic reaction cleanups- Columns or Phenol-chloroform extraction? - (Nov/15/2007 )
Is the recovery of DNA following a enzymatic reaction (eg. ligation, restriction enzyme digest, etc) efficient using commercial spin column kits (eg Qiaquick) compared to the phenol-chloroform extraction and alc ppt? I am thinking of ways of spending up a protocol but wonder if this will be at the expense of lower DNA yield.
Thanks so much for sharing your thoughts.
-timpanister-
After restriction enzyme digest or PCR, we usually run the DNA on gel and purify through a column. You have to anticipate a loss of 10% of DNA normally unless you have a bad column where you might lose most of the DNA. This usually happens with 1-2 columns in an entire kit.
After ligation, we transform the mixture so there is no need to purify DNA.
-scolix-
QUOTE (scolix @ Nov 16 2007, 03:41 PM)
After restriction enzyme digest or PCR, we usually run the DNA on gel and purify through a column. You have to anticipate a loss of 10% of DNA normally unless you have a bad column where you might lose most of the DNA. This usually happens with 1-2 columns in an entire kit.
After ligation, we transform the mixture so there is no need to purify DNA.
After ligation, we transform the mixture so there is no need to purify DNA.
I normally use these columns too for cleaning up reactions and gel extraction. There is an article on improving yields with these kits at bitesize bio.
And I also agree with Scolix that it is not necessary to purify a ligation - just go straight to the transformation.
-bitesizebio guy-