Daniel:Notebook/ComboLock/2017-3-13

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Bolt Test (Started Wednesday March 8)[edit]

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Protocol-No Ligase[edit]

Sample Matrix

Sample Condition
Sample 5 (AB) Normal
Sample 6 (AB) No Ligase
  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 following into a 0.2 mL tube
      1. 4 uL each PCCA-03 and PCCB-03 (10 uM each)
      2. 12 uL wash buffer
    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. Repeat wash step above

Protocol-Part 2-LB hybridization & ligation[edit]

  1. Bolt and Latch Hybridization/Ligation
    1. Set up the following master mix
    2. Reagent Stock Conc Final Conc./Amt uL added Master Mix (4.2X)
      T4 Ligase Reaction Buffer 5X 1X 4 16.8
      Latch Oligo 10 uM 10 pmol 1 4.2
      Lock Oligo 10 uM 10 pmol 1 4.2
      nfH2O NA NA 13 54.6
      Total     19 79.8
    3. Add 19 uL probe mix to each sample
    4. Incubate at 55C for 10 minutes
    5. Incubate at 25C for 10 minutes
    6. Lower the temperature to 20C; wait 30 seconds then take off incubator
    7. Add 1 uL T4 Ligase to each reaction
    8. Incubate at RT for 15 min
    9. Heat kill enzyme by incubating at 65C for 10 minutes
    10. Wash twice with 100 uL wash buffer
  2. Elution
    1. Add 50 uL wash buffer to each sample
    2. Cook at 95C for 15 minutes
  3. qPCR
    1. Make master mixes according to following recipes
    2. Reagent Single Rxn MMC1 (6.2X) MMC2 (6.2X) MMP (6.2X)
      Primer Pair NA p24/p4RC p6/p12RC p24/p12RC
      uL Fwd Primer (10 uM) 1 6.2 6.2 6.2
      uL Rev Primer (10 uM) 1 6.2 6.2 6.2
      uL nfH2O 21 130.2 130.2 130.2
      uL Kapa SYBR Fast 25 155 155 155
      Total 48 297.6 297.6 297.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-03132017-BoltTest-NoLigase.png
      • Note: The parentheses in the descriptions indicate which sample was used
      • Note:The colors are according to master mix, targeting either C1 (blue), C2 (red), or product (green)
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  4. TBE Gel
    1. Mix 360 uL TBE, 90 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 23 minutes at 230V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]

Results-CANTC and Efficiency[edit]

Take this with the Friday results

File:20170313-BoltTest-NoLigase-SampleValues.png

File:20170313-BoltTest-NoLigase-Efficiencies.png

The above graph (and Friday's, which is just this graph without the Sample-TR2 and No Ligase samples) had potentially wrong calculations for efficiency, since they only calculated the amount in the qPCR reaction (2 uL), which is a small fraction of the amount eluted (50 uL).

File:20170313-BoltTest-NoLigase-Efficiencies-Corrected.png