primer restriction sites

Ole Skovgaard olesk at RUC.DK
Fri Sep 1 09:46:18 EST 1995


In article <421s2f$dnc at mserv1.dl.ac.uk> Andrei Popov <ANDREI.POPOV at bbsrc.ac.uk> writes:
>From: Andrei Popov <ANDREI.POPOV at bbsrc.ac.uk>
>Subject: RE: primer restriction sites
>Date: 30 Aug 1995 15:19:59 +0100

>>Dear Colleagues,
>>        I designed a pair of primers which have restriction sites at
>>their 5' ends.  I did not add additional bp since I intended to clone
>>that into a PCR vector first.  I am wondering if I can directly digest
>>them with the enzymes for cloning into an expression vector.  I would
>>appreciate your help if you have done that for these two enzymes.
>>        The enzymes are: Bam HI and Hind III.
>>
>>Frank Chen
>>
>You can: remove primers,
>polish the ends (1U T4 pol, 50mkM dNTP 5-10 min at RT),
>co-ligate PCR  fragments, then digest with your RE (REs).

>regards

>Andrei
The New England Biolab catalog indicates that at least one and pref. two add. 
nt. are needed for BamHI. With HindIII its much worse. Even add. 3 nt. does 
not make the end into a good substrate, so Andrei´s or other approaches are 
needed.
good luck
ole




******************************************
****   Ole Skovgaard                  ****
****   Dept. Life. Sc. & Chemistry    ****
****   Roskilde University, DK        ****
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