Hi all,
I've designed a set of primers to evaluate gene expression with amplicon size of 100 bp. I checked the best annealing temperature in a conventional PCR (gradient), then seeing the best band in a agarose gel. I choose this temperature to use on qPCR (Sybr method) following the manufacturing instructions (Sybr, primers and cDNA concentration). I didn't give any good amplification curves.
So, my primers are working well on conventional PCR but on qPCR they don't work.
qPCR experts can help me?
Thank you
Hi,
so you issue is the curve shape correct? You might relax the cycling protocol a bit. In addition the Magnesium COnc may differ between the mixes which, together with the buffer composition might affect the annealing temp to be used. In addition did you check PCR efficiency by running a dilution series? An End point PCR does not give any indication if you have an optimal efficiency.
So to summarize I would propose to relax the protocol, recheck annealing Temp (e.g. Gradient) as well as PCR efficiency. Let me know if you need more infos
Good Luck
Sven
