library construction problem

Frederik Boernke boernke at
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


using Qiaex gel purification kit (Qiagen). 2.5 ul purified DNA were


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


through several freeze/thaw cycles. How to check whether the ligation



Or could be the packaging or plating?

Any hints will be appreciated.



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

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