We often sequence directly lambda phage DNA, without problems. It was not
so before, the big difference came with the ThermoSequenase kits and labeled
dideoxynucleotides. The template has to be in greater amount than usual
(i.e. more than what we use for sequencing PCR fragments or plasmids),
usually around 1-5 micrograms per 20-25 ul reaction. We take care to get
rid of the RNA by digestion with RNase, followed by Sephadex G-50 removal of
the ribonucleotides and other small undigested pieces of RNA.
Regards,
wagner yotov, Univ. of Montreal
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Thomas J. Stelick
Sent: 7 septembre, 1999 13:41
To: Recipients of ABRF List
Subject: dnaseq: lambda dna
How have people been sequencing lamdba DNA? I have had some success, but
not many samples to try it on. Any recommendations would be helpful.
Tom Stelick
TJS11@cornell.edu
BioResource Center, Cornell University
157 biotech bldg.
Ithaca, NY 14853
(607)254-4857
http://brcweb.bio.cornell.edu