Daniel:Notebook/ComboLock/2017-2-7

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Revision as of 21:15, 8 February 2017 by >Djacobse (→‎v2 Oligos-Round 3)
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v2 Oligos-Round 3

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Based on yesterday's conclusions, I've redesigned the experimental protocol to be more sensitive about temperature. The biotin-streptavidin bond is best kept under 70C. I'll use the v3 oligos for now, and try again with v2 if we have a problem. I'm also going to test ethanol precipitation post-exonuclease digestion. This is because exonucleases (and Amp ligase for that matter) do not properly degrade from simple heat inactivation (at least not in the short times we use). The added enzymes can potentially produce problems downstream, and the exonucleases in particular may be responsible for the RCA reactions showing weaker amplification than they should, since all the RCA product is single-stranded. So I'll use ethanol precipitation to extract only the DNA from the mix.

Sample Matrix and Workflow Diagram

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No C probes
Sample 4 (AB) No phosphate activation


Workflow Diagram

File:20170207-CL-Workflow.png

Workflow diagram for the experiment. This experiment tests both the reduced temperatures as well as using ethanol precipitation post-exonuclease digestion.

Protocol-Part 1

  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
    2. Reagent uL per Sample uL Total (Samples+NT) uL Total (No Phosphate) uL Total (No C Probe)
      PCCA-03 (10 uM) 1 4 2 0
      PCCB-03 (10 uM) 1 4 2*** 0
      Wash buffer 3 12 6 10
      Total 5 20 10 10
    3. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    4. Add 5 uL probe mixture to beads
    5. Incubate at 40C for 1 hour with agitation
    6. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    7. Repeat wash step above
    8. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant

Protocol-Phase 2

  1. Lock and Latch Hybridization/Ligation
    1. Set up the following master mix
    2. Reagent Stock Conc Final Conc./Amt uL added Master Mix (8.2X)
      T4 Ligase Reaction Buffer 5X 1X 4 32.8
      Latch Oligo 10 uM 10 pmol 1 8.2
      Lock Oligo 10 uM 10 pmol 1 8.2
      nfH2O NA NA 13 106.6
      Total     19 155.8
    3. Add 19 uL probe mix to each sample
    4. Incubate at 55C for 20 minutes
    5. Lower the temperature to 20C; wait 30 seconds then take off incubator
    6. Add 1 uL T4 Ligase to each reaction
    7. Incubate at RT for 30 min
    8. Heat kill enzyme by incubating at 65C for 10 minutes
    9. Wash twice with 100 uL wash buffer
  2. Padlock Hybridization
    1. Make the following 8.1X master mix (1X)
      1. 16.2 uL 10 uM padlock0501(2uL)
      2. 16.2 uL Amp Ligase 10X reaction buffer(2uL)
      3. 129.6 uL nfH2O
      4. (16uL)
    2. Resuspend sample in 20 uL padlock buffer
    3. Incubate at 37C for 30 minutes
  3. Circularization
    1. Prepare 8.5X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (8.5x) (uL)
      NAD+ 5 mM 40 nmol 8 68
      dNTP 1 mM 600 pmol 0.6 5.1
      Betaine 5 M 15 umol 3 25.5
      10X AmpLigase Buffer 10X 1X 2 17
      Amp Ligase 5 U/uL 10 U 2 17
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 27.2
      nf H2O     1.2 8.4
      Total     20 140
    3. Add 20 uL phusion mix to 20 uL reaction
    4. Incubate at 55C for 2 hours

Protocol-Part3

  1. Exonuclease Digestion
    1. Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1 hour at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Take 10 uL and set aside for future testing
    2. Prepare 8.2X Master mix according to table below
    3. Add 15 uL master mix to each tube
    4. Add sample to new reaction tubes according to following table
    5. Reagent uL Added Master Mix (8.2X)
      Template 3 0
      RCA Primer (10 uM) 2.5 20.5
      dNTP (1 mM) 0.8 6.56
      10X Buffer 2 16.4
      Phi29 1 0
      nfH2O 10.7 87.74
      Total 20 131.2
    6. Incubate at 30C for 6 hours
    7. Incubate at 65C for 10 minutes;Hold at 10C until next step
  3. Ethanol Precipitation
    1. Add 70 uL H2O
    2. Transfer to a 1.5 mL tube
    3. Add 300 uL 100% EtOH, 30 uL 3M NaAOc, 1 uL glycoblue
    4. Incubate overnight at -80C

    Continued tomorrow