In my experience, PCR samples can be tricky. They work best (for me) if the
PCR product is first cleaned up using a kit (such as Promega PCR Prep) to
remove remaining dNTPs, buffers, primers, etc. Alternately, the fragment can
be run out on an agarose gel, excised, and purified (be sure to resuspend in
water, not TE). Also of importance is the primer used. Sometimes the primer
used to generate a fragment is not suitable for sequencing. Some of my clients
have great success with PCR fragments, and some can't get anything at all.
Good luck--
Sheryl Christofferson
OMRF DNA Sequencing Facility
Oklahoma City, OK
sherylc@omrf.ouhsc.edu
susan fetics wrote:
> 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