We have been having great success sequencing PCR fragments on our ABI 377
after DNA purification by QIAquick spin columns and plasmids purified by
QIAprep spin coulmns. A key to success is accurate quantitation of
template. We use DAPI dye fluorescence. For Plasmid DNA and PCR > 500 bp,
we use 400 ng/reaction. For PCR products less than 500 bp, we use 25 fmole
per reaction. For a 300 bp fragment, this corresponsd to about 5 ng of DNA
per reaction. The reference for the dapi method is Aldridge, et al., Am.
J. Hum. Gen. 36:546-564, 1984.
David F. Bishop, Ph.D. | EMail:bishop at msvax.mssm.edu
Dept. of Human Genetics, Box 1203 |
Mount Sinai School of Medicine | Phone: (212) 241-6946
5th Avenue and 100th Street, NY,NY 10029 | FAX: (212) 360-1809
>Greetings and fond(-ish) felicitations to all and sundry!
> I wonder if you folks out there could offer a mere beginner a
>-We're just setting up a group sequencing facility ('just' being the
>operative word, the 377 is still sitting comfortably in its rather large
>box), and I understand that the template concentration is an important
>factor (amongst others), in getting good 'reads'. Can anyone recommend the
>best way of accurately quantitating the DNA conc.? (Almost) any advice
>would be appreciated.
> Many thanks!
> Lucas Bowler
>> Dept. Mol. Microbiol.
> Univ. Sussex