library construction problem (fwd)

Li Juan Fang 96527869 at CALLISTO.SI.USHERB.CA
Tue Mar 16 18:27:51 EST 1999



Hi, Ricky. 
 If you went to check your ligation, you can do your ligation rxn in
10-20ul, then take 1-5ul and run it in an agarose gel. You may not see the
band corresponding to the MW=vector+insert. If the ligation worked you can
see a smear or some other band besides your vector band and insert band. 
If your ligation did not work, you can only see your vector and insert on
gel.
Good luck. 
Li 
On 10 Mar 1999, Frederik Boernke wrote:

> Hi all,
> 
> 
> 
> I am about to construct a genomic library of Erwinia in Stratagene's
> 
> ZAP Express vector without success. DNA was isolated using standard
> 
> protocols and subsequently subjected to a partial Sau3A digest. The
> 
> fraction containing the desired fragment size was resolved on an agarose
> 
> gel and fragments ranging from about 3 to 10 kb were excised and
> 
> purified
> 
> using Qiaex gel purification kit (Qiagen). 2.5 ul purified DNA were
> 
> ligated
> 
> into BamHI/CIAP treated lambda arms (Stratagene) in a total volume of
> 
> 5 ul (1 ul vector, 2.5 ul DNA, 0.5 ul buffer, 0.5 ul 10 mM rATP, 0.5 ul
> 
> T4-ligase from boehringer's rapid ligation kit). 3.5 ul of the ligation
> 
> were packed into phages using Gigapack III gold packaging extract 
> 
> (Stratagene) according to the manufactures manual. A dilution series
> 
> was plated onto NZY agar plates and incubated at 37 deg..
> 
> What I see is a single blue plaque where I plated 1 ul of the packed
> 
> library (not very efficient :( ) no plaques on the other plates.
> 
> 
> 
> What went wrong?
> 
> 
> 
> Is it the ligation? The genomic DNA was stored at - 20 deg. and already
> 
> went
> 
> through several freeze/thaw cycles. How to check whether the ligation
> 
> was
> 
> efficient?
> 
> Or could be the packaging or plating?
> 
> Any hints will be appreciated.
> 
> 
> 
> Ricky
> 
> ******************************************************************
> 
> Frederik Boernke
> Research Group of  Molecular Plant Physiology
> Institute for Plant Genetics and Crop Plant Research (IPK)
> Corrensstr. 3
> 06466 Gatersleben
> Tel.  039482 -5 321
> Fax. 039482 -5 515
> http://www.ipk-gatersleben.de
> 
> 





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