No ligation of PCR product in pGemT-Easy

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Mon Jul 28 18:02:11 EST 2003


There is one basic thing you may have overlooked!  Did you phophorylate the 
primers (prior to PCR) or the amplified products before ligation?


Quoting Joe <appelgjj at sci.uovs.ac.za>:

> Hi, 
> 
> I've obtained several clones by means of differential display, where
> after I've reamplified the clones, with Taq SuperPak (Sigma-Aldrich), 
> cleaned the PCR product by means of GFX PCR DNA purification kit
> (Amersham Biosciences) and ligated the purified clones into pGemT-Easy
> (Promega) and transformed into JM109 cells.  The transformed cells
> were grown on LB + 50mg/ml Ampicillin.  I've got cells that grew,
> extracted plasmid again with a miniprep, and digested with EcoR1, but
> got no insertions back.  I then plated the transformed cells on
> Xgal/IPTG plates and everyone was blue - meaning no recombination
> occurred.  Can anybody please help?
> 
> Thank you in advance
> 
> Joe Appelgryn
> M.Sc student
> Dept. of Plant Sciences
> University of the Free State
> RSA
> 


-- 
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---



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