Daniel:Notebook/ComboLock/2016-12-15

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Revision as of 23:49, 15 December 2016 by >Djacobse (→‎Results)
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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.

  1. qPCR
    1. Make master mix according to following recipes
    2. Master Mix Primers Primer Target 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Master Mix
      MMC1 p24/p4* C1 230 9.2 9.2 193.2 MMC1
      MMC2 p24/p4* C1 230 9.2 9.2 193.2 MMC1
      MMCP p6/p12* C2 230 9.2 9.2 193.2 MMC2
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to table in the following section
    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

qPCR Sample Matrix

Sample Condition Primers Primer Target Fraction Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL) Master Mix
Sample 1A Sample p24/p4* C1 Supernatant-PreRCA A1 2 25 1 1 21 50 MMC1
Sample 1B Sample p24/p4* C1 Supernatant-PreRCA A2 2 25 1 1 21 50 MMC1
Sample 1A Sample p6/p12* C2 Supernatant-PreRCA A3 2 25 1 1 21 50 MMC2
Sample 1B Sample p6/p12* C2 Supernatant-PreRCA A4 2 25 1 1 21 50 MMC2
Sample 1A Sample p24/p12* Circular Product Supernatant-PreRCA A5 2 25 1 1 21 50 MMCP
Sample 1B Sample p24/p12* Circular Product Supernatant-PreRCA A6 2 25 1 1 21 50 MMCP
Sample 1A Sample p24/p4* C1 Beads-PreRCA C1 2 25 1 1 21 50 MMC1
Sample 1B Sample p24/p4* C1 Beads-PreRCA C2 2 25 1 1 21 50 MMC1
Sample 1A Sample p6/p12* C2 Beads-PreRCA C3 2 25 1 1 21 50 MMC2
Sample 1B Sample p6/p12* C2 Beads-PreRCA C4 2 25 1 1 21 50 MMC2
Sample 1A Sample p24/p12* Circular Product Beads-PreRCA C5 2 25 1 1 21 50 MMCP
Sample 1B Sample p24/p12* Circular Product Beads-PreRCA C6 2 25 1 1 21 50 MMCP
Sample 3A No C Probes p24/p4* C1 Supernatant-PreRCA F1 2 25 1 1 21 50 MMC1
Sample 3B No C Probes p24/p4* C1 Supernatant-PreRCA F2 2 25 1 1 21 50 MMC1
Sample 3A No C Probes p6/p12* C2 Supernatant-PreRCA F3 2 25 1 1 21 50 MMC2
Sample 3B No C Probes p6/p12* C2 Supernatant-PreRCA F4 2 25 1 1 21 50 MMC2
Sample 3A No C Probes p24/p12* Circular Product Supernatant-PreRCA F5 2 25 1 1 21 50 MMCP
Sample 3B No C Probes p24/p12* Circular Product Supernatant-PreRCA F6 2 25 1 1 21 50 MMCP
Sample 3A No C Probes p24/p4* C1 Beads-PreRCA H1 2 25 1 1 21 50 MMC1
Sample 3B No C Probes p24/p4* C1 Beads-PreRCA H2 2 25 1 1 21 50 MMC1
Sample 3A No C Probes p6/p12* C2 Beads-PreRCA H3 2 25 1 1 21 50 MMC2
Sample 3B No C Probes p6/p12* C2 Beads-PreRCA H4 2 25 1 1 21 50 MMC2
Sample 3A No C Probes p24/p12* Circular Product Beads-PreRCA H5 2 25 1 1 21 50 MMCP
Sample 3B No C Probes p24/p12* Circular Product Beads-PreRCA H6 2 25 1 1 21 50 MMCP
NTC qPCR Primers Only p24/p4* C1 NA A7 2 25 1 1 23 50 MMC1
NTC qPCR Primers Only p6/p12* C2 NA C7 2 25 1 1 23 50 MMC2
NTC qPCR Primers Only p24/p12* Circular Product NA F7 2 25 1 1 23 50 MMCP

Overnight Capture

So based on the results above, I'd say that the latch and padlock are not both finding their targets at the same time, which is leading to a situation where nothing works. So I'm going to repeat the experiment but with an overnight capture step that will give everything time to settle. I'll incubate at 60C overnight for capture (The Tm of the adapters is designed to 65 btw). Tomorrow I will add the phusion reaction (Phusion polymerase + Amp Ligase) for 2 hours without taking the samples off the incubator.

Protocol

  1. Template-Bead Binding
    1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
    4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. C Probe Hybridization
    1. Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
    2. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    3. Add 5 uL probe mixture to beads
    4. Incubate at 40C for 1 hour with agitation
    5. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    6. Repeat wash step above
    7. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
  3. Latch and Padlock Hybridization
    1. Add following to a fresh 0.2 mL tube
      1. 12.2 uL Latch0001, 10 uM stock
      2. 12.2 uL Padlock0201, 10 uM stock
      3. 12.2 uL Amp10X buffer
      4. 85.4 uL nfH2O
    2. Incubate at 60C overnight

    Continued tomorrow