cloning genomic DNA fragments

sarah boomer sarai at u.washington.edu
Tue Jul 19 14:33:31 EST 1994


In article <00981A26.A3A070E0.6 at vaxa.weeg.uiowa.edu>,
pzheng at VAXA.WEEG.UIOWA.EDU wrote:

> Dear bionetters:
> I am desperate,  would someone out there help me?
> I  had a lot of troubles in sucloing DNA fragments into pBluescript.  By
> using a
> cDNA probe to screen a EMBL 3 genomic library, I was able to get a
> positive clone. 
> By using different restriction enzymes, I was also able to generate
> DNA fragments
> of different sizes that can hybridize to the cDNA probe.  However, the
> troubles
> started from there.  When I tried to clone those fragments[blunt-
> ended as well as
> sticky-ended] none of them could be ligated into Bluescript[no
> recombinants on
> plants.  And I believed the DNA fragment isolation and ligation
> precedures had no
> problems.  Anyone ever had similar problems? Also, where can I get
> primers
> flanking the EMBL 3 cloning site?  Thank you for your help!  John
> Zheng.  
> PZheng at vaxa.weeg.uiowa.edu

Is the DNA you're trying to clone methylated?  If so, you may want to try
switching your E. coli strain to a methylation-restriction deficient
strain, such as Stratagene's SURE (tm) strain.



More information about the Methods mailing list