Daniel:Notebook/ComboLock/2017-2-13

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Revision as of 01:36, 14 February 2017 by >Djacobse (→‎Results)
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Beads Binding Test (Started Thursday 2-9)

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Data Analysis

  1. Data was analyzed by target (template,C1 probe, and C2 probe)
  2. For each of the three potential targets:
    1. Values were averaged across the two technical replicates according to plate specs
    2. Averaged CT values were subtracted from the CT value of the NTC corresponding to that plate and target
    3. If the value of the differential was <0 the value was set to 0

Results are plotted below for each target separately.

Results

Controls Table

Control Plate 1 CT Plate 2 CT Plate 3 CT Plate 4 CT Plate 5 CT Plate 6 CT
NTC-Template 28.34 25.8 27.7 26.64 25.39 26.32
NTC-C1 31.74 31.24 30.25 31.08 29.65 31.27
NTC-C2 32.15 30.51 31.86 27.3 30.63 31.32
Pos-Template N/A N/A N/A N/A N/A N/A
Pos-C1 N/A N/A N/A N/A N/A N/A
Pos-C2 N/A 38.28 N/A N/A 35.12 N/A

Discussion

Note: For notation purposes:

  • Fraction refers to the time points, as from a column purification experiment. These are discrete time course fractions. The fractions are (in chronological order): Template, C Probe, 1st Wash, 2nd Wash, 3rd Wash, 4th Wash, Elution.
  • Target refers to the target of the qPCR primers. The targets are Template, C1 Probe, and C2 Probe.
  • Yes, template is both a fraction and a target.

Template

The results for the template are fairly straightforward.

  • There is a considerable amount of template present in the post-C probe wash, which is actually explained in better detail by the experiment I have planned for tomorrow, and is also touched on by the positive control section. Also, the samples that had the template fraction (RT, No Template, 30C, 40C, and 50C) received "NA"s for the template fraction. I think this is because they are overloaded. Again, see the experiment tomorrow and positive control section below.
  • The wash steps don't really have any rhyme or reason to them, there is simply a dwindling amount of template in every wash.
  • The elution fraction best shows the results. All the washes below and incl. 50C have approximately the same amount of template. At higher temperatures the amount of template eluted in the wash decreases with increasing temperature, with the 90C wash having 1000X less than the RT control
  • The "No Template" control condition is adequately below NTC for most fractions, and only slightly above when supposedly present.

C Probes

Discussed as a team since they are the same.

  • C probes did not amplify above NTC (or <1 cycle) in the "template" fraction. This is good as C probe had not been added yet.
  • A significant portion of C probes are available post-binding
  • The washes all contain some C probe but there is no real temperature difference
  • As for the template target, the C probe binding shows some temperature dependence in the elution fraction. Up until around 70C the elution is basically the same, but there is a weak drop-off in the 80C and 90C fractions. Note that the 90C fraction is ~5 cycles fewer, which is on the order of 30 times less available C probe than in the lower temperature washes
  • The No C Probe condition shows no significant amplification (whew*)

Positive Control