June Eva Paciga
jpaciga at com1.med.usf.edu
Mon Mar 3 18:28:05 EST 1997
Xin Wei wrote:
> Dear Netter: I have some difficulty with the Telomerase TRAP assay: My
> results just looks like kind of reverse from my expected, there are 4~6
> bands show in buffer control lane and not many bands present in positive
> sample lane. Actually I was very careful about PCR contamination, I used
> separate pipetman and hot start, it doesnÕt help for the bands in buffer
> control. I increase the Taq polymerase 2~3 times, it did raise the bands
> in positive control, but I am still concern the unspecific amplification
> by doing this. Could somebody give me some suggestion about these?
> Xin Wei
Are you using OnCor's TRAPeze Telomerase Detection Kit or your own
assay set up?
(OnCor's detection kit is pretty good for telomerase detection.(no
However, read on...
I would recommend diluting your extracts. If you have too much
template the primers and Taq polymerase become limiting. That is
probably why you see an increase in the product in your positive control
when you increase the Taq polymerase. I have used the OnCor TRAPeze kit
and I obtained a great ladder with cell extract from the equivalent of
1000 or 100 cells. However with the extract equivalent of 100,000 cells
I could not obtain a ladder.
As for the bands in the buffer only tube I would guess that these are
the formation of primer dimers. Do you see these bands in the
heat-inavtivated controls? Apparently this primer dimer formation is a
problem with the TRAP assay in general. Recently, I used Boehringer
Mannheim's Telomerase PCR ELISA Kit to detect and quantitate telomerase
activity and it is G R E A T!!!! Positive controls are very positive
and negative controls are truly negative. Again, I was able to detect
activity with extract equivalent of 100 cells and I could have diluted
the extract even further and detected activity. I read the absorbance
at 450 nm on a spectrophotometer using micro cuvettes that hold 100
microliters. The kit is designed so that the absorbance can be read
with a microplate ELISA reader however the actual numbers I recorded
were ten fold lower because the pathlength of a plate well is shorter.
NOTE: I am also not affiliated with Boehringer Mannheim either.
Both kits use non-isotopic detection.Although the OnCor kit can be used
with isotope I obtained good results staining the gel with SYBR GREEN
I. I hope this helps you. If I can help any further e-mail me.
June Eva Paciga
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