3700 sequencing

Leviten, Dina dleviten at ICOS.com
Fri May 12 10:40:32 EST 2000

I have seen 2 reasons for what you describe.  The first is that perhaps
there are salts competing when injecting--this would mean that all their
samples tail off.  To fix this, a EtOH precipitation usually does the trick.
For example, we ran tests on different mini-prep kits and found that Promega
Wizard Plus preps don't sequence well until EtOH ppt'd, however, the Promega
Wizard Plus SV preps work fine without the extra step.  (Qiagen work fine).
The second is if you only see a couple of their samples fail, while the
others look great--this usually means that the polymer load on that run for
that capillary probably had a bubble, these samples when directly re-run
(even on the same capillary) usually give great results.

Good luck!

Dina Leviten
Manager, DNA Microchemistry
ICOS, Corp.
22021 20th Avenue SE
Bothell, WA 98021
e-mail:  dleviten at icos.com
phone: (425)485-1900 x4402(lab) x5339(desk)
fax:  (425)485-1961

> -----Original Message-----
> From:	Stuart Bayliss [SMTP:stuart.bayliss at csc.mrc.ac.uk]
> Sent:	Friday, May 12, 2000 1:36 AM
> To:	Recipients of ABRF List
> Cc:	autoseq
> Subject:	3700 sequencing
> Hi,
> I have recently begun to use a 3700 for sequencing in a core facility.
> Certain users have noticed samples in which data quiality tails off quite
> early (200-300 base region).  These users usually obtain >650 bases when
> the samples are run on a 377.
> I was thinking it may be due to the electrokinetic injection
> preferentially
> injecting salts etc into the capillary ahead of the sample in turn leading
> to low signal/longer fragments missing...which maybe wouldn't cause undue
> problems on a gel system.
> Has anyone seen such cases?  I'd appreciate any help you could give.
> Regards,
> Stuart.
> Stuart Bayliss,
> Services Co-ordinator,
> Genetics Core Facility,
> MRC Clinical Science Centre,
> Clinical Research Building,
> Hammersmith Hospital,
> Du Cane Road,
> London.
> W12 0NN
> email:  stuart.bayliss at csc.mrc.ac.uk
> Tel (office):   0208 383 8305
> Tel (lab):      0208 383 3181
> Fax:            0208 383 8338

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