Daniel:Notebook/ComboLock/2017-2-14

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Revision as of 21:05, 14 February 2017 by >Djacobse (→‎C Probe Binding Efficiency)
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Beads Binding Test (Started Thursday 2-9)[edit]

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Positive Control Test[edit]

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.

  1. qPCR
    1. Make qPCR master mixes according to following recipes
    2. Master Mix Recipes
      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
    3. Add 48 uL of appropriate master mix and 2 uL sample according to plate layout
    4. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x45
      7. 72C 2 min
      8. 16C hold

Plate Layout[edit]

File:PlateLayout-20170214.png

Note: Blank indicates lanes that were measured but had nothing (even master mix) in them.

Results[edit]

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

File:PosControlDilutions-20170214.png

Discussion[edit]

So what this tells me is that at the 350 ng range (taking direct from a 10 uM stock) is WAY too much. Even for the C2 amplification, which did have a CT, the CT was basically gibberish. This was probably caused by too much template getting in the way of primer binding. At approximately 1000X less (350 pg) this worked fine for all samples. A 100X dilution (3.5 ng) was fine for the C probes but failed for template. This is good to keep in mind when including positive controls in the future. This also explains several other times I have used positive controls and failed in previous experiments. Including yesterday.

So, looking at yesterday's results in the new lens, we can make a few more conclusions:

  • It now makes perfect sense why the template fraction always failed. From the calculation in yesterday's discussion, there is way more template than needed to bind to the 2 uL beads that I use. So there is still around 90% template in solution, which corresponds to 315 ng in my experiment, which will always fail.
  • This also means the C probe binds efficiently, since the fraction was reduced to a measurable amount in the supernatant of the C probe fraction. See below.

C Probe Binding Efficiency[edit]

Data Analysis Pipeline

  1. Calculate average value of the samples for a given technical replicate
  2. Calculate Cycles above NTC using the NTC for that target (C1 or C2) on that specific run (Plates 4-6 or PosCon)
  3. Calculate the fold amplification over the 35 pg sample from PosCon (for C1 or C2)
  4. The amount in the wash was therefore estimated as 35 pg * the fold amplification (final result given in ng)
  5. Since 10 pmol C probe each was added, this is ~180 ng; each sample was calculated for percent in supernatant and the estimated percent bound (100-% in supernatant)

The table in the next section shows (some) of the intermediate values and final values. The graph is a scatter of the cycles above NTC and the % bound. For clarity, the "No C Probe" conditions are not shown (they are 0,0 and therefore outliers).


File:20170210-BeadsqPCR-CProbeBindingEfficency.png

C Probe Binding Results Table[edit]

Name Average CT Abs(Difference) Cycles Above NTC Fold Amplification to 35 pg Est. Amt in Wash (ng) Estimated Fraction in Supernatant (%) Estimated Fraction Bound (%)
Room Temp-C Probe fraction (C1) 7.36 0.22 22.9 200.9 7.0 3.9 96.1
No Template-C Probe fraction (C1) 7.25 0.08 23.0 216.8 7.6 4.2 95.8
30C Wash-C Probe fraction (C1) 7.23 0.19 23.0 220.6 7.7 4.3 95.7
40C Wash-C Probe fraction (C1) 7.60 0.3 22.7 170.1 6.0 3.3 96.7
50C Wash-C Probe fraction (C1) 7.15 0.12 23.1 232.3 8.1 4.5 95.5
60C Wash-C Probe fraction (C1) 6.89 0.32 24.2 494.6 17.3 9.6 90.4
70C Wash-C Probe fraction (C1) 7.03 0.08 24.1 448.8 15.7 8.7 91.3
80C Wash-C Probe fraction (C1) 7.00 0.03 24.1 459.8 16.1 8.9 91.1
90C Wash-C Probe fraction (C1) 7.16 0.07 23.9 411.6 14.4 8.0 92.0
No C Probe-C Probe fraction (C1) 30.43 0.55 0.7 4.1 1.4 7.9 0.0
Room Temp-C Probe fraction (C2) 7.34 0.09 23.3 164.3 5.7 3.2 96.8
No Template-C Probe fraction (C2) 7.76 0.77 22.9 122.8 4.3 2.4 97.6
30C Wash-C Probe fraction (C2) 7.86 0.86 22.8 114.2 4.0 2.2 97.8
40C Wash-C Probe fraction (C2) 7.35 0.19 23.3 163.1 5.7 3.2 96.8
50C Wash-C Probe fraction (C2) 7.20 0.02 23.4 180.4 6.3 3.5 96.5
60C Wash-C Probe fraction (C2) 7.36 0.24 24.0 260.5 9.1 5.1 94.9
70C Wash-C Probe fraction (C2) 7.25 0.14 24.1 281.1 9.8 5.5 94.5
80C Wash-C Probe fraction (C2) 7.26 0.03 24.1 280.1 9.8 5.4 94.6
90C Wash-C Probe fraction (C2) 7.43 0.17 23.9 249.0 8.7 4.8 95.2
No C Probe-C Probe fraction (C2) 31.14 0.11 0.2 1.8 6.4 3.5 0.0