[Automated-sequencing] Re: variation in libraries (subject line
Phillip San Miguel
pmiguel at purdue.edu
Thu Sep 22 09:55:14 EST 2005
Fulton, Ciaran wrote:
> Hi Philip...if you reinoculate further DWP plates overnight from your
> glycerol stocks...
> <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>.
> What volume would you transfer to the fresh media?
Either pin innoculation is used (we use the Genetix 384-well disposable
pin tools) or about 10ul gets transfered using an old Biomek 2K robot.
If you are have problems with growth, the latter is recommended. The pin
tool transfers less than a ul.
Cultures for miniprep are grown overnight on a rotary shaker (300-400
rpm) at 37 oC. Of course you need a decent shaker that won't splash the
cultures across wells. We also cover the cultures with qiagen airpore
If you are in 96-well deeps, I would advise against overgrowth. Although
I haven't seen the "broad peak issue" with DH10Bs, in other host strains
(eg TOP10), it was worse in overgrown cultures. I should add that was
using qiagen REAL preps. REAL preps are just alkaline lysis, lysate
clearing through a filter plate and precipitation of the cleared lysate
with isopropanol. We use Eppendorf 96 or 384-well preps now. These
entail a binding to a glass-filter matrix in the presence of chaotrope
followed by washing and elution.
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