How to cut PCR product with restriction enzyme ?

Bernard Heymann bheymann at bragg.bio.purdue.edu
Fri May 27 11:51:58 EST 1994


In article <2s497j$s38 at mserv1.dl.ac.uk>, Wirote Tuntiwechapikul
<mdbcs001 at cmu.chiangmai.ac.th> wrote:

> 	May anyone tell me how to cut the PCR product with restriction enzyme?
> Recently I have performed the PCR of a gene and then I want to detect the 
> point mutation in the product with a restriction enzyme. I have tried to 
> cut it directly from the mixture but there are some problems. First, I 
> used the reaction mixture of 50 microlitre and it was too much to be loaded 
> in the slab gel for further detection by electrophoresis. So I applied 
> half of them instead and found that the band was almost unperceivable and 
> also it was not cut completely. Do I have to extract the PCR product out 
> of the reaction mixture before cutting with the restriction enzyme ? I 
> really do not want to do  that because it takes time and may lose some 
> product.
> 	The second question is how to adjust the reaction volume for 
> restriction enzyme cutting . In the definition of restriction enzyme, one 
> unit of restriction enzyme is the efficacy of cutting the DNA of 1 
> microgram in 50 microliter in one hours. If I want to cut 1 microgram of 
> DNA in 20 microliter, how can I do ?
> 	Thank you in advance for all the respond and suggestion.
> 					Wirote.

Hi Wirote

After trying a few different combinations of steps in cloning PCR products,
I settled on the following:

First I make sure I have good synthesis of the fragment by running 5 or 10
ul on a gel. Then I purify the fragment from the rest of the reaction
mixture using Promega's PCR prep and eluting my fragment into 40 ul water.
It is important to purify your fragment away from the polymerase since it
can fill in overhangs and add A's to blunt ends after restriction. From 10
to 20 ul of the purified product is then digested with 2 to 5 units of
enzyme overnight (I persistently failed to clone with shorter digestion
times). Finally I ligate the fragment into the vector with about a five
times excess, also overnight at 4 C.

This protocol has worked for me almost every time, but I'd like to hear
other people's comments on this.


-- 
Bernard Heymann
bheymann at bragg.bio.purdue.edu



More information about the Methods mailing list