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Template conc.

David F. Bishop bishop at msvax.mssm.edu
Wed Mar 20 08:46:42 EST 1996


Dear Lucas,

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.

Cheers,

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
>little advice?
>-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





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