Daniel:Notebook/ComboLock/2017-2-14
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Beads Binding Test (Started Thursday 2-9)
Positive Control Test
Yesterday's results showed that the positive controls failed time and time again. Looking at the math again, by adding 2 uL 10 uM template to the positive control reactions, I was adding >100X the amount Kapa's protocol recommends. So this time I ran a dilution series that covers each of the positive controls in dilutions of undiluted to 1:10000.
- qPCR
- Make qPCR master mixes according to following recipes Master Mix Recipes
- Add 48 uL of appropriate master mix and 2 uL sample according to plate layout
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x45
- 72C 2 min
- 16C hold
Master Mix | Primer Pair | Master Mix NoX | Forward Primer Amt (10 uM) | Reverse Primer Amt (10 uM) | uL nfH2O | uL 2X Kapa SYBR Fast | Total |
MMT | PCCT-F/PCCT-R | 11.2 | 11.2 | 11.2 | 235.2 | 280 | 537.6 |
MMC1 | primer24/primer4RC | 11.2 | 11.2 | 11.2 | 235.2 | 280 | 537.6 |
MMC2 | primer6/primer12RC | 11.2 | 11.2 | 11.2 | 235.2 | 280 | 537.6 |