we prefer PCR fragment sequencing over the analysis of a number of clones.
The only point which is really important is to get rid of the PCR primers=
-
which is obvious. Given that, our users are happy with direct PCR fragmen=
t
sequence data which is informative for heterozygotes and related stuff. W=
e
get the template DNA from the users (and had to train them over time). To
get acceptable success rate with the still very heterogenous quality of t=
he
template DNA, we use a 50%-BD term reaction as standard.
Best regards, Bernd
>Hi Susan
>
>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 y=
our
>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 cyc=
les
>> in 4uL halfterm (Genpak) and 2uL BigDye (ABI), centrifuge thru priceto=
n
>> sep
>> columns, dry the samples, denature samples at 90 degrees C for 3 minut=
es
>> 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
>>
_____________________________________________________________________
PD Dr. Bernd Weisshaar
MPI fuer Zuechtungsforschung
Carl-von-Linn=E9-Weg 10
50829 Koeln
Germany
Tel. office: +49-221-5062 590 Tel. lab: +49-221-5062 311
Fax: +49-221-5062 313
mailto:weisshaa@mpiz-koeln.mpg.de
WWW-URL: http://www.mpiz-koeln.mpg.de/~weisshaa/BW-info.html