Daniel:Notebook/ComboLock/2016-12-15

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Revision as of 19:00, 15 December 2016 by >Djacobse (→‎C Probe and Product Detection)
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Mirror Probe Test (Started Tuesday)

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RCA on Beads/Supernatant Fractions

  1. qPCR
    1. Make master mix according to following recipe
      1. 298.2 uL nfH2O
      2. 355 uL SYBR Fast
      3. 14.2 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section
    4. Sample Condition Fraction Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 1A Sample Supernatant A1 20 2 25 1 1 21 50
      Sample 1B Sample Supernatant A2 20 2 25 1 1 21 50
      Sample 2A No Template Supernatant A3 21 2 25 1 1 21 50
      Sample 2B No Template Supernatant A4 21 2 25 1 1 21 50
      Sample 3A No C Probe Supernatant A5 22 2 25 1 1 21 50
      Sample 3B No C Probe Supernatant A6 22 2 25 1 1 21 50
      Sample 1A Sample Beads H1 23 2 25 1 1 21 50
      Sample 1B Sample Beads H2 23 2 25 1 1 21 50
      Sample 2A No Template Beads H3 24 2 25 1 1 21 50
      Sample 2B No Template Beads H4 24 2 25 1 1 21 50
      Sample 3A No C Probe Beads H5 25 2 25 1 1 21 50
      Sample 3B No C Probe Beads H6 25 2 25 1 1 21 50
      NTC qPCR Primers Only NA A7 26 2 25 1 1 23 50
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix 144 uL TBE, 36 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results

Although there is a slight smear in the region of correct size (211bp), the smear is present in every lane (except the NTC). This suggests the smear is a result of the beads in general and not really related to the product. My conclusion is that the circular product never formed.

C Probe and Product Detection

I'm now going to investigate the presence/absence of C probes and circularized products using the adapter primers.


qPCR Sample Matrix

Sample Condition Primers Primer Target Fraction Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
Sample 1A Sample p24/p4* C1 Supernatant-PreRCA A1 20 2 25 1 1 21 50
Sample 1B Sample p24/p4* C1 Supernatant-PreRCA A2 20 2 25 1 1 21 50
Sample 1A Sample p6/p12* C2 Supernatant-PreRCA A3 21 2 25 1 1 21 50
Sample 1B Sample p6/p12* C2 Supernatant-PreRCA A4 21 2 25 1 1 21 50
Sample 1A Sample p24/p12* Circular Product Supernatant-PreRCA A3 21 2 25 1 1 21 50
Sample 1B Sample p24/p12* Circular Product Supernatant-PreRCA A4 21 2 25 1 1 21 50
Sample 1A Sample p24/p4* C1 Beads-PreRCA A1 20 2 25 1 1 21 50
Sample 1B Sample p24/p4* C1 Beads-PreRCA A2 20 2 25 1 1 21 50
Sample 1A Sample p6/p12* C2 Beads-PreRCA A3 21 2 25 1 1 21 50
Sample 1B Sample p6/p12* C2 Beads-PreRCA A4 21 2 25 1 1 21 50
Sample 1A Sample p24/p12* Circular Product Beads-PreRCA A3 21 2 25 1 1 21 50
Sample 1B Sample p24/p12* Circular Product Beads-PreRCA A4 21 2 25 1 1 21 50
Sample 3A No C Probes p24/p4* C1 Supernatant-PreRCA A1 20 2 25 1 1 21 50
Sample 3B No C Probes p24/p4* C1 Supernatant-PreRCA A2 20 2 25 1 1 21 50
Sample 3A No C Probes p6/p12* C2 Supernatant-PreRCA A3 21 2 25 1 1 21 50
Sample 3B No C Probes p6/p12* C2 Supernatant-PreRCA A4 21 2 25 1 1 21 50
Sample 3A No C Probes p24/p12* Circular Product Supernatant-PreRCA A3 21 2 25 1 1 21 50
Sample 3B No C Probes p24/p12* Circular Product Supernatant-PreRCA A4 21 2 25 1 1 21 50
Sample 3A No C Probes p24/p4* C1 Beads-PreRCA A1 20 2 25 1 1 21 50
Sample 3B No C Probes p24/p4* C1 Beads-PreRCA A2 20 2 25 1 1 21 50
Sample 3A No C Probes p6/p12* C2 Beads-PreRCA A3 21 2 25 1 1 21 50
Sample 3B No C Probes p6/p12* C2 Beads-PreRCA A4 21 2 25 1 1 21 50
Sample 3A No C Probes p24/p12* Circular Product Beads-PreRCA A3 21 2 25 1 1 21 50
Sample 3B No C Probes p24/p12* Circular Product Beads-PreRCA A4 21 2 25 1 1 21 50
NTC qPCR Primers Only p24/p4* C1 NA A7 26 2 25 1 1 23 50
NTC qPCR Primers Only p6/p12* C2 NA A7 26 2 25 1 1 23 50
NTC qPCR Primers Only p24/p12* Circular Product NA A7 26 2 25 1 1 23 50