If the person is adding too much template you would see high signal
strength right in the beginning of the sequence followed by a
significant drop off to weak and noisy sequence. If the person is
certain of the template and primers, he is probably losing his
sample during the purification. Precipitation has high sample loss
in comparison to other purification methods. Two alternatives and
an overall suggestion are as follows:
1. Run a control with the samples. This will verify whether it is
a template/primer issue or a purification issue. If the control
is fine then it is a template/primer issue. If the control
fails
then it is a matter of the purification.
3. For a purification issue, purify using a column. The person
will reduce sample loss and increase signal strength.
Overall suggestion --> To minimize time and effort, run a control
and purify using a column.
Jennifer Brosius
Sequencing Technologist
ValiGen
300 Pheasant Run
Newtown, PA 18940
Phone:(215)504-4444
Fax:(215)504-1546
http://www.Valigen.net
-----Original Message-----
From: sfetics@server.nybc.org [mailto:sfetics@server.nybc.org]
Sent: Friday, June 30, 2000 4:51 PM
To: Recipients of ABRF List
Subject: DNA Seq
I have a client who is having difficulty getting sequences out of his
samples on my 373XL. He's using Lambda-phage DNA which is approximately
40
Kb in size. His vector is lambda-phage, and he is trying to sequence the
insert. He first tried an internal primer, but saw no sequence, then
tried
a universal primer, and still there was no sequence. He's been
precipitating with ethanol. Because of the large size, instead of
submitting the normal .5µg amount, he's been submitting 1.0 - 2.0 µg.
Should he be submitting smaller quantities, such as .5µg? If anyone can
help with some possible solutions, I would greatly appreciate it.
___________________________________________________________________
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
For information about the MicroChemistry lab at NYBC or for request
forms,
please visit:
http://www.nybloodcenter.org/framesets/FS-3C17.htm
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