library construction problem

Frederik Boernke boernke at nospam.ipk-gatersleben.de
Wed Mar 10 04:19:50 EST 1999


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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