qPCR - Test Young Lab qPCR Calibration (Repeat)

This is a repeat of an earlier run from today, but with a different qPCR plate. [Here’re master mix calcs (bottom half of page)(https://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20110204-01.jpg). Cycling params were as follows:

  • 95C - 10min

40 Cycles of:

  • 95C - 15s

  • 55C - 30s

  • 72C - 1m

Melt curve.


All samples amplified and showed a proper dissociation curve. However, it does look like there’s a spread of ~3 Cts across the plate. This is not good, as this is the equivalent of ~10-fold difference in gene expression. Will repeat again and see if specific wells show the same relative differences in Cts.