Hi out there
I recently had six primer pairs synthesised by Oswel, UK. Three of them
were
labelled with the ABI-dye FAM, two with TET and one with HEX. Primers
are
stored in tubes and dilutions as they were received from the
manufacturer. We then make a dilution (with water) and mix of primers to
a final concentration of 12,5 uM. This stock is stored at 4oC and used
for PCR (e.g. 1ul to 50ul PCR rxn).
After a few days, extra non-PCR dependent peaks starts to develop as
shown
below when PCR products are analysed on a ABI PRISM 310 Genetic
Analyser:
FAM labelled primers develop a =91blue=92 peak at 130 bp
TET labelled primers develop a =91green=92 peak at 140 bp
HEX labelled primer develops a =91green=92 peak at 140 bp and a =91yellow=
=92
peak at
180 bp
All six primers develop these extra bands dependent of their label. The
undiluted stock stored at -20oC does not show these extra bands
(although
the FAM primers have a faint blue peak at 130 bp) The non-specific
band
interferes with alleles and makes genotyping hell.
I have never seen these curious extra bands even after storage of
diluted
primers for several months. Has anybody else seen this before and/or
does
anybody have an explantion or suggestions as to how I solve this problem
of extra peaks?
Regards,
Flemming
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If you want to reply to me please remove
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Flemming Larsen
Dept. of Clin. Immunol., sect. 7631
National University Hospital
Tagensvej 20
DK-2200 Copenhagen N
Denmark
phone: +45 35 45 78 49
fax: +45 31 39 87 66
