Daniel:Notebook/ComboLock/2017-2-2

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RCA Time Test (Started Friday Jan 27)[edit]

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Phosphorothiorate RCA Primer (Started Yesterday)[edit]

  1. qPCR
    1. Make qPCR master mix according to following recipe
      1. 361.2 uL nfH2O
      2. 17.2 uL AmpF6.4
      3. 430 uL Kapa SYBR Fast
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL AmpR indexed according to table
    4. Sample Sample Condition AmpR Index Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 1AN Sample 3 hours 20 A8 2 25 1 1 21 50
      Sample 1BN Sample 3 hours 20 A7 2 25 1 1 21 50
      Sample 3AN No C Probe 3 hours 21 A6 2 25 1 1 21 50
      Sample 3BN No C Probe 3 hours 21 A5 2 25 1 1 21 50
      Sample 1AO Sample 3 hours-Epi 22 A4 2 25 1 1 21 50
      Sample 1BO Sample 3 hours-Epi 22 A3 2 25 1 1 21 50
      Sample 3AO No C Probe 3 hours-Epi 23 A2 2 25 1 1 21 50
      Sample 3BO No C Probe 3 hours-Epi 23 A1 2 25 1 1 21 50
      NTC     24 H1 0 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 94 uL TBE, 24 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]

Version 2 Oligos Round 2[edit]

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Since I've been having so much trouble with the version 3 oligos, I want to test the v2 oligos again.

Sample Matrix[edit]

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

Protocol-Phase 1[edit]

  1. Phosphorylation-MirrorC Probe B (PCCB-02)
    1. Set up reaction according to table
    2. Reagent Stock Conc Final Conc./Amount uL added
      T4 Polynucleotide Kinase Buffer 10X 1X 2
      ATP 10 mM 1 mM 2
      PCCB-02 10 uM 100 pmol total 10
      T4 DNA Kinase 10 U/uL 10 U 1
      nfH2O NA NA 5
      Total     20
    3. Incubate at 37C for 30 min
    4. Heat kill enzyme with 15 minutes at 65C
  2. 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
  3. C Probe Hybridization
    1. Combine following into a 0.2 mL tube
    2. Reagent uL per Sample uL Total
      PCCA 1 6
      Mirror C Probe B reaction 2 12
      Wash buffer 2 12
      Total 5 30
    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; start part 4 after incubation begins
    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[edit]

  4. Phosphorylation-Lock oligo
    1. Set up reaction according to table
    2. Reagent Stock Conc Final Conc./Amount uL added
      T4 DNA Ligase Buffer 10X 1X 2
      ATP 10 mM 1 mM 2
      Lock0201 10 uM 10 pmol total 10
      T4 DNA Kinase 10 U/uL 10 U 1
      nfH2O NA NA 5
      Total     20
    3. Incubate at 37C for 30 min
    4. Heat kill enzyme with 15 minutes at 65C
  5. Lock and Latch Hybridization/Ligation
    1. Set up the following master mix; Note: for sample 4 add un-phosphorylated Lock0201
    2. Reagent Stock Conc Final Conc./Amt uL added Master Mix (6.2X) Master Mix (2.2X)
      T4 Ligase Reaction Buffer 5X 1X 4 24.8 8.8
      Latch Oligo 10 uM 10 pmol 1 6.2 2.2
      Lock Oligo Phosphate Reaction (5 uM Lock) 10 pmol 2 12.4 *2.2*
      nfH2O NA NA 12 86.8 39.2
      Total     19 74.4 26.4

      This table had errors. Correct table below but I used the table above

      Reagent Stock Conc Final Conc./Amt uL added Master Mix (6.2X) Master Mix (2.2X)
      T4 Ligase Reaction Buffer 5X 1X 4 24.8 8.8
      Latch Oligo 10 uM 10 pmol 1 6.2 2.2
      Lock Oligo Phosphate Reaction (5 uM Lock) 10 pmol 2 12.4 *2.2*
      nfH2O NA NA 12 74.4 28.6
      Total     19 117.8 39.6
    3. Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201
    4. Incubate at 95C for 5 min
    5. Lower temperature to 40C; incubate 20 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 20 min
    9. Heat kill enzyme by incubating at 65C for 10 minutes
    10. Wash twice with 100 uL wash buffer

Protocol-Part 3-Padlock Hybridization and Circularization[edit]

  1. Padlock Hybridization
    1. Make the following master mix
      1. 16.4 uL 10 uM Padlock0401A
      2. 16.4 uL Amp Ligase 10X reaction buffer
      3. 131.2 uL nfH2O
    2. Resuspend sample in 20 uL padlock buffer
    3. Incubate at 37C for 10 minutes
  2. 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 40C for 4 hours
    5. Heat kill enzyme by incubating 10 minutes at 95C
    6. Hold overnight at 12C

    Continued tomorrow

    Buffers[edit]

    Wash Buffer

    Reagent Stock Final Dilution Amt in 20 mL
    NaCl 1.5 M 0.5 M 3 6.66 mL
    Tris-HCl 500 mM 20 mM 25 800 uL
    EDTA 0.5M 1 mM 500 40 uL
    nf H2O NA NA NA 12.5 mL