Daniel:Notebook/ComboLock/2017-2-13
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Beads Binding Test (Started Thursday 2-9)[edit]
Data Analysis[edit]
- Data was analyzed by target (template,C1 probe, and C2 probe)
- For each of the three potential targets:
- Values were averaged across the two technical replicates according to plate specs
- Averaged CT values were subtracted from the CT value of the NTC corresponding to that plate and target
- If the value of the differential was <0 the value was set to 0
Results are plotted below for each target separately.
Results[edit]
- Note for the Y axis that this is the number of cycles less than the NTC (or NTC-sample). This means the greater the number the more target DNA present
- BeadsExperiment-20170210-Template-Annotated.png
Elution curves-template
- BeadsExperiment-20170210-C1Averages-Annotated.png
Elution curves-C1 probe
- BeadsExperiment-20170210-C2Averages-Annotated.png
Elution curves-C2 probe
- 20170210-BeadsqPCR-ElutionFraction.png
Elution fraction as a function of temperature. Fraction is relative to the available amount for RT sample.
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[edit]
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*)
- Interestingly the No Template condition is around the same amplification as the 80C in the elution fraction. Since there is no template to bind to the C probes should just wash out but they do hang around. Either they stick to the beads or are just floating in solution I could never quite get rid of
Positive Control
- The positive controls did not amplify, on the whole. Two plates managed amplification with CTs well above NTC level. Chris and I theorize there might be too much template.
- The NEB beads protocol recommends 25 uL of 8 pmol/uL biotinylated Poly dT per 125 uL beads. This is 200 pmol biotin/125 uL beads=1.6 pmol biotin/uL beads. I add 20 pmol biotinylated oligo per 2 uL bead (10 pmol/uL bead). This is ~10X more than the suggested protocol, which would result in (probably) a lot of free oligo left in solution
- The Kapa protocol recommends (for SYBR FAST) <1 ng/uL. For the positive controls I added 2 uL of 10 pmol/uL, which with 54bp template ends up around 300 ng/uL. So that's at least two orders of magnitude too high. Tomorrow I'll do a dilution experiment to confirm and discuss the impact of the results on these results.