Daniel:Notebook/ComboLock/2017-3-10: Difference between revisions

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<li>Cycle differences were averaged when technical replicates existed; e.g. for all experimental samples</li></ol>
<li>Cycle differences were averaged when technical replicates existed; e.g. for all experimental samples</li></ol>
<li>Quantitation and Efficiency</li>
<li>Quantitation and Efficiency</li>
<li>Positive control values were calculated from IDT dilutions (2 uL of 10 nM = 0.02 pmol) or Qubit measurements<li>
<ol type="A">
<li>Positive control values were calculated from IDT dilutions (2 uL of 10 nM = 0.02 pmol) or Qubit measurements</li>
<li>Qubit measurements (product)</li>
<li>Qubit measurements (product)</li>
<ol type="a">
<ol type="a">

Revision as of 00:17, 11 March 2017

Bolt Test (Started Wednesday)

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Data Analysis

  1. Normalization
    1. CT values were obtained from the qPCR curves via the Biorad qPCR results
    2. CT values were subtracted from either the NTC CT value or positive control CT value (for quantitation)
    3. Cycle differences were averaged when technical replicates existed; e.g. for all experimental samples
  2. Quantitation and Efficiency
    1. Positive control values were calculated from IDT dilutions (2 uL of 10 nM = 0.02 pmol) or Qubit measurements
    2. Qubit measurements (product)
      1. 3100, 3210, and 2810 ng/mL
      2. Divide by 100X (Qubit measured sample was 100X qPCR template)
      3. Convert to pmol in 2 uL: 1.81*10^-3, 1.87*10^-3, 1.64*10^-3 pmol
      4. Average: 1.8*10-3 pmol
    3. Amount in the sample is 2^(cycle difference poscon-sample) * 1.8*10^-3 pmol
    4. From bead math, ~3.2 pmol of template should bind to bead, which means 3.2 pmol of C probe may bind and 3.2 pmol of ligation product may form, the theoretical maximum assuming 100% bead binding, 100% C probe binding, 100% wash efficiency, and 100% ligation efficiency

Results

Cycle Thresholds-Sample Presence

File:20170310-BoltTest-SampleValues.png

  • The results from the samples alone are varied
    • Off-target amplification is low. Off target is primers targeting products that are not present; specifically C1 primers for C2, C2 primers for C1, and product primers for C1 or C2.
    • The No C Probe negative control is most effective as a negative control (see below as to why the others are not). This makes sense as neither C probe nor the product should be able to amplify without the C probes
    • The No Template control has product. The product amplified in 8 cycles over NTC, which is 16 cycles slower than the sample's product, which indicates the efficiency without a template is ~65000X less than with the template. Since the template only acts as a binding hub for the C probes and consequently latch/bolt, it is theoretically possible to have the complete reaction entirely in solution. This does indicate, however, that the wash steps are not completely effective
    • The No phosphate (PO4) control is still somewhat effective. This is the most concerning, since ligation should not occur without a 5' phosphate. The phosphate product showed in 6-7 cycles less than sample, which indicates only a ~100X dropoff in efficiency without phosphate. Most literature I've seen indicates it should be much worse. But I recommend doing a NO LIGASE sample to repeat the experiment. Without ligase no product should form. I will perform this on Monday since I don't have time today.

Quantitation and Efficiency

File:20170310-BoltTest-EfficiencyBeads.png