[Automated-sequencing] Re: Sequences informations

Phillip San Miguel pmiguel at purdue.edu
Tue Feb 28 11:01:35 EST 2006


attaoua at montp.inserm.fr wrote:
> Dear Sir,
> Iwant to thank you for your Email.
> My sequence measures 2500 bp. Currently I'm using a machine for sequencing
> "Beckman CEQ 8000". I'm sequencing now regions in genes.
>               Sincerly yours.
>              Redha ATTAOUA
>              IURC -Montpellier-
> 

2500 bp should work with Beckman's standard protocol.

Is your template DNA from a PCR reaction?

If so, you will need to "clean-up" the PCR reaction--most important here 
is to get rid of the PCR primers. If they  are present in your 
sequencing reaction, then they will both generate sequence products. The 
result will be two sequences mixed together--unreadable. Companies such 
as Qiagen have PCR reaction clean-up columns that will work for this 
purpose.

If you are using an agarose gel to clean-up your PCR reaction, do not 
use TBE buffer. Use another buffer, like TAE. I have been told that the 
borate interferes with the sequencing reaction.

Phillip SanMiguel
Purdue Genomics Core Facility



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