Loosing my genomic DNA clones - what a pitty!!!

neuschaefer at gsf.de neuschaefer at gsf.de
Wed Nov 20 04:47:17 EST 2002

Hy, boardies!
For several months now I am cloning genomic sequences from Arabidopsis to enlarge our microarray chip. Moast technical things worked fine, getting almost all genes cloned, but now after several generations, I am loosing inserts as well as total plasmids. After a sequencing session with 130 new cloned inserts, it came out that I lost 17 % percent of formerly cloned genes (positive by gene specific primed PCR!!!), and all this is going further and further. 
What did I wrong? Did anyone had a comparable problem? Did I collect resistent, but insert-free plasmids? 
O.K. this could not be excluded, but in this quantity. I am not a beginner in that field.  

The result of my experience: After screening a clone positve, IMMEDIATELY make a miniprep. to have a reservoir of positive cloned material. DON´T TRUST a stock culture. 

Thanks for your ideas,

Olaf Neuschaefer-Rube, Ph.D.
Institute for biochemical plant pathology - 
phytodiagnostics group
GSF - research center for environmet and health
Ingolstaedeter Landstrasse 1
D-85764 Neuherberg



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