Daniel:Notebook/ComboLock/2017-3-10
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Bolt Test (Started Wednesday)
Data Analysis
- Normalization
- CT values were obtained from the qPCR curves via the Biorad qPCR results
- CT values were subtracted from either the NTC CT value or positive control CT value (for quantitation)
- Cycle differences were averaged when technical replicates existed; e.g. for all experimental samples
- Quantitation and Efficiency
- Positive control values were calculated from IDT dilutions (2 uL of 10 nM = 0.02 pmol) or Qubit measurements
- Qubit measurements (product)
- 3100, 3210, and 2810 ng/mL
- Divide by 100X (Qubit measured sample was 100X qPCR template)
- Convert to pmol in 2 uL: 1.81*10^-3, 1.87*10^-3, 1.64*10^-3 pmol
- Average: 1.8*10-3 pmol
- Amount in the sample is 2^(cycle difference poscon-sample) * 1.8*10^-3 pmol
- 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.