Posted 14 May 2013 - 04:09 AM
Please review the attached photo and let me know if the first three primers are good enough for qPCR (PECAM-1, VEcad, and ACTA2). I designed these primers on NCBI's primerBlast and also enabled the option to search for unintended targets, in which only one target returned (no unintended targets).
The picture below the PCR was ran at 35 cycles with a 1 min. extension time. My amplicons are less than 200 bp though and for qPCR my extension time will be 30 seconds. The point I'm trying to get at is whether the products seen below 172 for PECAM-1, around 600 for VEcad, and around 800 for ACTA2 are from adjustable parameters in the PCR or from poor primer design.
Thank you for any insight. Some details: 10 ng cDNA Template, 0.2 uM For & Rev primer concentration
Posted 14 May 2013 - 12:37 PM
Edit: If you are using software to measure the relative density of each band, it may be alright. You could get a rough estimate, but I would personally try altering your methods first.
Edited by jerryshelly1, 14 May 2013 - 12:39 PM.
Posted 15 May 2013 - 06:34 PM