PCR Problem

julia juliam at ariel.ucs.unimelb.edu.au
Tue Jul 11 02:01:23 EST 1995


My name is Julia.  I am undertaking my PhD at the Peter Maccallum 
Cancer Institute in Melbourne Australia.  I am using PCR to amplify
 mouse DNA using a variety of SSLP's obtained from the Whitehead
 Institute.  I am writing for help because all of a sudden my PCR has
 gone from working beautifully to half working to not working at all.  Here is my procedure.  Please if anyone can give me any clues that would be great.  

Because I am dealing with many tumour DNA samples at once,  the 
procedure I use is to make up a PCR mix.  This contains everything
 except the DNA.  That is the primers, both unlabelled and labelled,
 MgCl2, dNTP's, taq polymerase, polymerase buffer and H2O.  I add the
 DNA to the PCR mix.  I then place the samples into the PCR machine 
and do a 5 min. denature at 94C before starting the amplification 
cycles.  Usually what I see on a PAGE/formamide gel are 2 bands per
 DNA sample since the DNA was isolated from heterozygous mice. 
 Occassionally I will see loss of heterozygosity which is what I am
looking for.  Now what I am seeing on these gels is one band only. 
 Bizarre! It appears that the primers are preferentially annealing to 
and amplifying one allele.  However,  when I run the 2 genomic DNA 
controls I get amplification of one of them, the same one that
amplified in the tumour  DNA samples but there is no amplification of
 the other DNA control. What am I to make of this? Has something
 happened to the DNA? Should I try a hot start? I await your reply. 

Julia McKenzie
 juliam at ariel.ucs.melbuni.edu.au

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