Bizzare digestion pattern

Eric J Hebert Eric at bioch.tamu.edu
Fri Jun 17 17:32:19 EST 1994


In article <2ts6kc$qc at mserv1.dl.ac.uk> "GWDGV1::KCHOWDH" <kchowdh at gwdgv1.dnet.gwdg.de> writes:
>Path: news.tamu.edu!cs.utexas.edu!swrinde!pipex!uknet!daresbury!not-for-mail
>From: "GWDGV1::KCHOWDH" <kchowdh at gwdgv1.dnet.gwdg.de>
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: RE: Bizzare digestion pattern
>Date: 17 Jun 1994 13:57:48 +0100
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>Sender: daemon at mserv1.dl.ac.uk
>Distribution: bionet
>Message-ID: <2ts6kc$qc at mserv1.dl.ac.uk>
>Original-To: methods at dl.ac.uk


>Hi,
>Are you quite sure about the orientation of the 600 bp plasmid?
>Is it possible that this clone has the insert in the other orientation such 
>that both the RI and BstEII sites are next to each other? Just a quick thought. 
>Kamal Chowdhury.

This is still a possibility, I would recheck to determine this is not the case.


[lots of stuff deleted]

Second,  why overexpress each of the small fragments in pUC19, if the two 
products have compatiable sequences why not use a PCR step (overlap extension 
method) to make the complete DNA product for example.  I am assuming here that 
the two fragments constitute some larger gene made of individual fragments 
that you have synthesized or modified chemically or enzymatically.

DNA product 1

5'--------------------------------------------------BstEII-3'

DNA product 2
5'-BstEII--------------------------------------------3'

These two in PCR or possible a Klenow reaction following denaturation and 
reannealing could yield

DNA product 3 (1 + 2)

5'---------------------BstEII----------------------3'

If you do not understand what I mean ask and I can explain in more detail and 
give you several references for such techniques.  Of course if the 3' end of 
DNA 1 and the 5' of DNA II are not complementary then it will never work.

Just a thought.

Good Luck and let us know how things work out.

E


erich at bioch.tamu.edu



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