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

Wolfgang Schechinger hubahopp at gmx.de
Wed Nov 20 12:41:30 EST 2002

Dear Olaf, 

Bugs that can survive with a shorter plasmid (or even integrate the
resistance gene into their genome) have some advatage in growth, esp when
you use high copy number vectors.
In my experience, glycerol stocks loose activty after some time in the
freezer, too.

I think that your conclusion is very important. Even when you only have one
single insert and not a library, it makes sense to miniprep and put that
material in several safe places. To start a maxiprep from a purified
plasmid just takes 2 days. One just should do the same what one (should)
do(es) with important computer data: Making backups. DNA is data, too, in
some sense :-) 

To your loss of variety problem:
Make simple set of calculations. In every generation (I mean approx 30 min)
only 99.99 or 99.9 or just 99 % of your bacteria do what you what them to
do. Then, how much will you have left after say a total of 24h  or 48 hrs?

0.99    ^ 48 = 61%    (38%) (24 hrs -> 48 generations, in brackes the
result for 96 generations) 
0.999   ^ 48 = 95%    (91%)
0.9999  ^ 48 = 99.5%  (99%)

Reads aaargh to me. Maybe explains some the garbage one sometimes sees when
analyzing minipreps.



At 09:47 AM 2002/11/20 -0000, neuschaefer at gsf.de wrote:
>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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