AW: Amersham ET terminators for 3700

Glenis Wiebe wiebe at mpi-cbg.de
Mon Oct 13 10:50:22 EST 2003


Hi Verena,

We regularly use 3 min. extensions with BigDye v3.1, and are still able
to collect around 1000 bases at >99% accuracy. (phred20>800)

Glenis

-- 
Glenis Wiebe
Facility Leader, DNA Sequencing
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 Dresden, Germany

Tel: +49-351-210-2628
Fax: +49-351-210-2000
e-mail: wiebe at mpi-cbg.de
http://www.mpi-cbg.de


Verena Bohle wrote:
> Hello everyone, hello Phillip,
>
> if you reduce the amount of BigDye to 1/16th, do you reduce the amount of D=
> NA in the same proportion?
>
> Has anyone tried to reduce the extension time using BigDye? What did you no=
> tice then?
>
> Thank you for any comments
> Verena Bohle
>
>
>
> -----Urspr=FCngliche Nachricht-----
> Von:=09Phillip San Miguel [SMTP:pmiguel at purdue.edu]
> Gesendet am:=09Samstag, 27. September 2003 19:02
> An:=09autoseq at net.bio.net
> Betreff:=09Re: Amersham ET terminators for 3700
>
> Paul,
>
> =09I have, but never tried below 1/2 reactions. Amersham's polymerase/chemi=
> stry
> allows much quicker thermal cycling (extensions are only 1min instead of 4 =
> min).
> But I think their polymerase is not as thermal stable. So while BD runs fin=
> e for
> 99 cycles--at least in the presence of 5% DMSO, I think the Amersham chemis=
> try
> will not.
>
> =09Also BD v3.1 under version 2.0 of data collection in our hands just give=
> s
> better results than Amersham's chemistry.
>
> =09If you are looking to save money, why not drop to 1/16th reactions with =
> the Big
> Dye--that works fine.
>
> Phillip SanMiguel
> Purdue Genomics Core Facility
>
> Paul Shinn wrote:
>
>>   Does anyone use Amersham ET Terminators for sequencing plasmid on the
>>3700?  We've been using BigDye for years but I can save a considerable
>>amount if I can switch to the ET Terminators.  I need to dilute them to
>>1/6rxn to save any money (I do 1/8 BD now).  Can you dilute it this low
>>for the 3700?  Are you happy with the quality?
>>
>>=09=09=09=09Thanks, Paul
>>
>>
>>---
>>Paul Shinn                   pshinn at neomorph.salk.edu
>>SIGnAL                       http://signal.salk.edu
>>Sequencing Coordinator       http://genome.salk.edu
>>(858) 453-4100 ext 1796
>>
>
>
> ---
>
> ---
>
>



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