We regularly use 3 min. extensions with BigDye v3.1, and are still able
to collect around 1000 bases at >99% accuracy. (phred20>800)
Facility Leader, DNA Sequencing
Max Planck Institute of Molecular Cell Biology and Genetics
01307 Dresden, Germany
e-mail: wiebe at mpi-cbg.dehttp://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
>> =09I have, but never tried below 1/2 reactions. Amersham's polymerase/chemi=
> allows much quicker thermal cycling (extensions are only 1min instead of 4 =
> 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=
> will not.
>> =09Also BD v3.1 under version 2.0 of data collection in our hands just give=
> 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?
>>Paul Shinn pshinn at neomorph.salk.edu>>SIGnAL http://signal.salk.edu>>Sequencing Coordinator http://genome.salk.edu>>(858) 453-4100 ext 1796