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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