Recently, the Young Lab’s ABI 7300 qPCR machine was calibrated. Steven asked me to run a plate and see how well the calibration worked. Ran a plate with C.gigas gDNA and Gigas 18s primers (SR ID: 156 and 157) that are known to amplify gDNA. [Master mix calcs are here (top 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 - 30s
Melt curve.
Results:
Absolutely no amplification of any kind. However, I did use one of our conventional PCR plates and not one of the ABI “prism” plates. Additionally, when I removed the plate from the machine, the plate looked as though it had been vigorously shaken:
Will repeat this qPCR with a proper ABI “prism” plate.