[Automated-sequencing] Re: variation in libraries was: Autoseq Digest, Vol 4, Issue 5 - Email found in subject

Phillip San Miguel pmiguel at purdue.edu
Thu Sep 22 09:30:16 EST 2005


Richard Cooke wrote:
> Phillip San Miguel wrote:
> 
> 
>>Duncan Clark wrote:
>>
>>
>>>Historians believe that in newspost
>>><mailman.683.1127317265.29381.autoseq at net.bio.net> on Wed, 21 Sep
>>>2005, Phillip San Miguel <pmiguel at purdue.edu> penned the following
>>>literary masterpiece:
>>>
>>>
>>>>If, however, you are using the strains that Invitrogen normally
>>>>includes with it's cloning kits--those give us enough trouble that
>>>>we toss them and buy DH10B electrocompetant cells.
>>>
>>>
>>>
>>>Which strains don't work for you?
>>>
>>>Duncan
>>
>>I stick with DH10B, DH5alpha and the XL's where I can. Many other
>>strains caused this issue (although a long time ago.) Top10 has given
>>me the broad peak issue.
>>
>>But to tell the truth, this might have only happen with a miniprep
>>method we no longer use...
>>
>>Phillip
>>
>>_______________________________________________
> 
> 
> Having had problems with different competent cells and/or miniprep
> methods, I (and, I presume, many others) would be interested to have
> feedback on what does and doesn't work for capillary sequencing. We do a
> lot of sequencing work for colleagues and I'd really like to be able to
> give clear guidelines on what to use. Could you share your experience?
> 
> Richard
> 
Ahh, well. In a nutshell, anything that results higher salt and/or glop 
in the final reactions that get loaded on the machine well tend to 
produce less robust results. Salt competes with sequence products for 
electrokinetic injection and "glop" seems correlated with a "broad peak 
phenomenon".

The host strain DH10B works well for us. We try to always use it for 
high throughput sequencing.

It's a complex issue though and "your milage may vary".

Phillip



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