RE: DNA Seq

Leviten, Dina (dleviten@icos.com)
Thu, 17 Jun 1999 08:50:53 -0700

Susan,
We've had great luck with PCR samples. We ask people to clean them up with
Qiaquick (Qiagen), then quantitate them as accurately as possible, then
submit them. We were using the 373XL, and would use amounts of PCR product
based on size. (eg. 200bp use 50-100ng, 1-3Kb use 125-250ng). We used the
regular recommeded sequencing PCR protocol and cleaned up with spin columns.
We'd get complete sequence including the big stop peaks at the end of the
fragment. I hope this works for you!
Dina Leviten
ICOS, Corp

> -----Original Message-----
> From: sfetics@server.nybc.org [SMTP:sfetics@server.nybc.org]
> Sent: Wednesday, June 16, 1999 7:28 AM
> To: Recipients of ABRF List
> Subject: DNA Seq
>
> hi-
> i'm new at dna sequencing. i use the PE 373XL, 64 lanes. i am getting no
> sequences from PCR samples. my protocol for all samples is: PCR 25 cycles
> in 4uL halfterm (Genpak) and 2uL BigDye (ABI), centrifuge thru priceton
> sep
> columns, dry the samples, denature samples at 90 degrees C for 3 minutes
> in
> 5 uL EDTA and deionized formamide, then i load the samples in the comb.
> should i be treating the pcr samples differnt than the other template
> samples? my other samples (DNA) and all the controls work fine, except for
> a few misses. but i am getting blank lanes any time i try to sequence pcr
> samples.
>
> thanks!
>
> -susan
> ___________________________________________________________________
>
> Susan K Fetics
> Laboratory of MicroChemistry
> Lindsley F. Kimball Research Institute
> The New York Blood Center
> 310 E. 67th St., 3rd floor
> New York, NY 10021
>
> phone: (212) 570-3188
> sfetics@nybc.org