Removal of PCR inhibitors
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Nov 24 10:47:07 EST 1999
In article <383bddbc.49782822 at news.netspace.net.au>,
jkcarson at netspace.net.au writes
>If we use a 9ul volume of template DNA prepared using the guanidinium
>method we do not get any PCR product but if we use a 1ul volume as
>template the PCR works. We have concluded that an inhibitor is
>co-extracting. BSA does not neutralise the effect.
SDS lysis of cells? I presume you do an ethanol or methanol based wash
of the diatoms to get rid of the guanidine prior to eluting with water
or TE? Are you acid washing the diatoms prior to use or autoclaving them
What will mess up the PCR is SDS, ethanol/methanol or guanidine traces
left behind and eluting with the water.
If using SDS try substituting Triton X-100 as per recent Indian lab's
Analytical Biochem paper - the author has posted in this newsgroup
recently so check the archives.
Do extra washes.
Dry the diatoms better to drive off the ethanol/methanol.
Primarily elute with water - not TE. Reason 9ul of TE with 1mM EDTA in a
20ul PCR will chelate out 0.675mM Mg of your 1.5mM Mg. Adding in extra
Mg would compensate or maybe eluting with TE (0.1mM EDTA). Alternatively
Let us all know what the answer is.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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