[Automated-sequencing] Re: variation in libraries (subject line edited)

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? 
>  
> Ciaran



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 
adhesive sheets.

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.


Phillip



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