I've been working on generating qPCR standards in plasmid form. I have used the pCR4.1 Topo-TA system and verified a few clones using PCR for the presence of my insert (the two fragments I've cloned are 200 bp and 110 bp), the bands are nice and bright and correctly sized.
When I do a test qPCR run using SYBR green, I have no amplification. A positive control set of standards which used another template was successfully amplified. I also tried cleaning up the plasmids using precipitation to no avail. I have attached the conventional PCR, there are four clones for each insert and two negative controls.
Any idea what I have overlooked? Thanks to anyone who can help!
2X SYBR green
10 umoles each primer
in 20 ul reaction
95 2 min
Edited by janaki, 02 July 2014 - 07:41 AM.