Daniel:Notebook/ComboLock/2017-2-14

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Revision as of 19:55, 14 February 2017 by >Djacobse (→‎Plate Layout)
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Beads Binding Test (Started Thursday 2-9)

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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.

  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

File:PlateLayout-20170214.png

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 0.180000000000001 19.8
Template-35 pg 12 13 10.66 0.0700000000000003 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 0.140000000000001 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 0.0199999999999996 19.9
C2-35 pg 37 38 14.98 0.129999999999999 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]]