Restriction digestion of PCR fragments

Sailesh Surapureddi SaiSu at MCB.LiU.SE
Fri Aug 18 14:11:18 EST 1995


>To: methods-and-reagents at net.bio.net
>From: akravetz at Walden.MO.NET (Andy Kravetz)
>Subject: Re: Restriction digestion of PCR fragments
>Date: 18 Aug 1995 18:24:13 GMT
>
>B VISSER * x2818 Plantkunde * (pbv at rs.uovs.ac.za) wrote:
>: I am in the process of digesting amplified cDNA fragments with Not1 of which 
>: a restriction site was incoporated into the primers.  I am not able to fully 
>: digest the fragments, even though sufficient nucleotides are present on the 
>: 5' side of the restriction site.  Could Ethidium bromide play a role in the 
>: inhibition of the digestion of the DNA?  I usually run the fragments on a 
>: prep gel and cut the desired fragments from the gel with phenol/chloroform 
>: which is followed by a ethanol precipitation.  Could this be the reason?
>
>I am having the same type of problem as you. I am trying to cut the ends 
>of a 400bp fragment amplified so I can use it in a ligation. It appears, 
>dispite having extra bases that it is not working well. I am considering 
>running it on LMP and then just cutting it in the agarose, then doing the 
>ligation in the agarose. If I do this, will I have any problems and can 
>anyone else see a problem with this. Thanks


Hi,
	This is to both of your quries, what exactly is the buffer you are trying to 
use for the restriction digestion, and is it a double digestion.
	If you could let me know this, then probably I could give a recepie for another    
buffer which could be used for sucessful digestion. 
	You may contact me either on this news group or directly.
Sailesh.



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