primer restriction sites

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

In article <421s2f$dnc at> Andrei Popov <ANDREI.POPOV at> writes:
>From: Andrei Popov <ANDREI.POPOV at>
>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).


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 
good luck

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

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