[Automated-sequencing] Re: variation in libraries

Phillip San Miguel pmiguel at purdue.edu
Tue Sep 20 10:48:13 EST 2005


Fulton, Ciaran wrote:
> Hi...I was wondering if anyone has had experience of variable length
> of read statistics within and between different 96-well plates for
> specific cDNA libraries. We used to pick manually into 96 DWP but
> increasing throughput meant we moved over to a QPix. The results
> haven't been great with it but we need to optimise it. But when we
> went back to picking by hand the results were awful. We normally
> generate the cDNA libraries using the Invitrogen CloneMiner kit and
> electroporate into DH10B cells. However, another transformation
> attempt at the same library by heat shock and into DH5-alpha cells
> has meant a massive drop in yield across the plate. The LOR results
> are so variable between plates that we are wrecking our heads trying
> to come up with answers. Done al the usual yield increasing steps we
> can think of...Terrific broth, increasing time etc, but no luck. Any
> suggestions
> 
> Dr. Ciaran Fulton Senior Scientist

Are you seeing variability in your DNA yields? How about cell density 
after growth (size of pellets)?

Are the "length of read" variabilies the result of low signal strength 
or mixed signal (double sequence) or something else?

Picking from colonies on agar into deep well plates with a Qpix can be 
tricky. The plates need to be very evenly filled. Otherwise either the 
tips don't actually touch the liquid culture medium or they go too deep 
and your pins can become contaminated.

We almost always pick with our Qpix into short plates containing liquid 
media + antibiotic and 7% glycerol (vol/vol). These just grow overnight 
without shaking and serve as glycerol stocks. From there pin 
innoculation or pippetting can be used to innoculate deep well plates.

-- 
Phillip SanMiguel
Purdue Genomics Core Facility



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