Help with PCR
Scott Wildenberg DGM-IHG
wilden at lenti.med.umn.edu
Wed Sep 14 18:53:45 EST 1994
I am writing to see if anyone has any suggestions or advice on the
following PCR problem:
I have several human fibroblast cell lines that I have extracted DNA
from using our lab's standard DNA extraction protocol (We extract from
blood all of the time). Two of the cell lines are from patients with a
disorder we are studying, and the remaining cell lines are from "normal"
controls.
I have not been able to amplify this DNA. I have tried
reextracting the DNA to clean it up, adding different amounts of DNA to
the reaction, increasing and decreasing Mg concentration, increasing the
amount of Taq in the reaction, and amplifying with different primers. In
all cases only my positive control is amplified.
Recently I mixed positive control DNA with the sample DNA in the
ratios 4:1, 2:1, 1:1, 1:2, and 1:4. All of these do amplify, suggesting
that there is nothing in the solution tha is inhibitin the reaction.
Furthermore in one of those reactions I have 50 ng of positive control
DNA (out of a total of 200ng). The band I get in that reaction is
similar in intensity to a reaction where the only DNA is 40ng od posotive
control DNA suggesting that there is nothing in the positive control that
promotes amplification.
All of this leads me to think that there is something about the
DNA itself that is the problem. Since I have several samples from normal
individuals, I have a hard time believing that they could all be missing
a part of the genome. Has anyone ever experienced anything like this or
have any suggestions? Also let me know if you think I am overlooking
anything.
Thanks for the help. Sorry about the lenghty message.
Scott C. Wildenberg
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Division of Genetics and Metabolism Voice (612) 625-5628
University of Minnesota Fax (612) 624-6645
Box 485 UMHC Email wilden at gene.med.umn.edu
Harvard Street at East River Road
Minneapolis, MN 55455
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