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
