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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