To be on the safe side, our lab tend not to sequence PCR samples! They are
known to be very awkward. Most people prefer to clone them first
(blunt-ended vector) and then sequence.
If you really need to sequence the PCR samples, then it would be better to
try using 8ul of the BD terminator kit instead of 2ul. Lower volume
reactions should only really be used for 'easy' stuff. Cleanliness of your
sample is also critical for PCR sequencing.
Kind Regards
Andrew
_______________________________________________________________
AstraZeneca R & D Charnwood
Molecular Biology, Bakewell Road, Loughborough, Leics, ENGLAND LE11 5RH
Tel: +44 (0)1509 644213 Mobile: +44 (0)778 8595040 Fax: +44 (0)1509
645557
andrew.walding@charnwood.gb.astra.com
> ----------
> From: sfetics@server.nybc.org[SMTP:sfetics@server.nybc.org]
> Sent: 16 June 1999 15:27
> 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
>