Daniel:Notebook/ComboLock/2017-2-14: Difference between revisions
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Revision as of 19:58, 14 February 2017
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 |
Plate Layout
Note: Blank indicates lanes that were measured but had nothing (even master mix) in them.
Results
Averaged CT Values and Differential Table
Name | Well 1 | Well 2 | Average CT | Abs(Difference) | Cycles Above NTC |
Template-350 ng | 2 | 3 | NA | NA | NA |
Template-35 ng | 4 | 5 | NA | NA | NA |
Template-3.5 ng | 6 | 7 | NA | NA | NA |
Template-350 pg | 8 | 9 | 6.81 | 0.18 | 19.8 |
Template-35 pg | 12 | 13 | 10.66 | 0.07 | 16.0 |
C1-350 ng | 14 | 15 | NA | NA | NA |
C1-35 ng | 16 | 17 | NA | NA | NA |
C1-3.5 ng | 18 | 19 | 7.53 | 0.2 | 22.8 |
C1-350 pg | 23 | 24 | 12.04 | 1.24 | 18.3 |
C1-35 pg | 25 | 26 | 15.10 | 0.14 | 15.2 |
C2-350 ng | 27 | 28 | 37.81 | 0.32 | |
C2-35 ng | 29 | 30 | 5.75 | 0.37 | 25.2 |
C2-3.5 ng | 33 | 34 | 7.72 | 0.12 | 23.2 |
C2-350 pg | 35 | 36 | 11.02 | 0.02 | 19.9 |
C2-35 pg | 37 | 38 | 14.98 | 0.13 | 15.9 |
NTC-Template | 10 | 10 | 26.61 | 0 | |
NTC-C1 | 11 | 11 | 30.34 | 0 | |
NTC-C2 | 20 | 20 | 30.91 | 0 |
Graph
[[Image:|600px]]