ABI-labelled primers develop extra peaks
flar at biobase.delete.dk
Wed Apr 1 10:52:49 EST 1998
Hi out there
I recently had six primer pairs synthesised by Oswel, UK. Three of them
labelled with the ABI-dye FAM, two with TET and one with HEX. Primers
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
below when PCR products are analysed on a ABI PRISM 310 Genetic
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=
All six primers develop these extra bands dependent of their label. The
undiluted stock stored at -20oC does not show these extra bands
the FAM primers have a faint blue peak at 130 bp) The non-specific
interferes with alleles and makes genotyping hell.
I have never seen these curious extra bands even after storage of
primers for several months. Has anybody else seen this before and/or
anybody have an explantion or suggestions as to how I solve this problem
of extra peaks?
If you want to reply to me please remove
the .delet form my email address.
Dept. of Clin. Immunol., sect. 7631
National University Hospital
DK-2200 Copenhagen N
phone: +45 35 45 78 49
fax: +45 31 39 87 66
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