I am using millipore kit (TRAPeze Kit) to determine telomerase activity. I am getting non-specific multiple bands in the negative control (only CHAPS lysis buffer).
Is this common? Are there any steps to eliminate non-specific amplification?
Article Telomerase Recruitment Requires both TCAB1 and Cajal Bodies ...
I think it is due to primer-dimer or contamination of PCR products in CHAPS lysis buffer. I recommend to use new CHAPS lysis buffer.
I used Wege et al 2003 and just a standard ABI SYBR green master mix with good results. I got more negative control signal when using the primers in a 1:1 ratio. Check the Wege paper for fine tuning
PMID: 12527792
I used the protocol with the following PMID: 19381925. Just be careful, there are a few typos in the paper. Be very careful with all your reagents; the hTERT complex contains RNA and so you need to be mindful to avoid RNAse degradation. If you do use this protocol, run the big gel at 250 V (make the volume up to 50 ml with dH2O, not add 50 ml dH2O) and I wouldn't recommend freezing/thawing any reagent/material more than four times. Best of luck whatever you choose!
Can You somehow show the picture of Chaps buffer lane (negative control)? If You want by e-mail
Just try with different lesser concentrations of MgCl2 in your reaction buffer and also reduce your primer concentrations..all the best..
ScienCell recently developed a qPCR-based, non-radioactive kit for measuring telomerase activity. The entire procedure can be done in one day, with hands-on time less than 2 hours. The primers' qPCR efficiency is close to 100%, making accurate quantification of telomerase activity possible. For more information, please go to:
https://www.sciencellonline.com/PS/8928.pdf
ScienCell also offered a few telomere length quantification by qPCR kits for human, mouse and rat.
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