Restriction digestion of PCR fragments
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
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.
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