Rekha,

A couple of questions - are you using cDNA from a cell type that you know expresses TERT? ESCs and EC cells and other stem cells should produce TERT. Have you tried amplifying any house keeping genes from the same cDNA pool?

One thing you need to keep in mind while designing primers is to make sure that the primers flank an intron. This is to ensure that you can distinguish amplification from cDNA as opposed to genomic contamination in the cDNA prep. I checked your primers against the genomic database. It amplifies a 215 bp fragment from both cDNA and genomic DNA, meaning that, if the primer works you can not be sure if your amplification is from expressed sequence or genomic DNA. This is especially important in cases such as TERT that you can potentially obtain false positive (if there is genomic contamination in the prep). I have had good luck with the following primers that amplifies a 298 bp product from EST and 1657 bp from genomic. Or look for other published primers. Even then, make sure that you do a blast search and check the size of amplicons from BOTH the EST and genomic DNA. I have seen that many published primers do not conform to this aspect. Also, in certain instances you may also want to verify whether a particular EST has pseudogenes, prime examples being OCT4 and NANOG. This can complicate your analysis. Well, this is a long answer for a small problem. But hope this helps.

Forward - 5'-GCTTCCTCAGGAACACCAAGA-3'

Reverse - 5'-TGCAACTTGCTCCAGACACTC-3'

Ta for this pair is 58 C