Daniel:Notebook/ComboLock/2016-8-17

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Revision as of 22:47, 17 August 2016 by >Djacobse (→‎Protocol)
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Positive Control Amplicon Production (Started yesterday)

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Protocol

  1. Size select gel
    1. Mix 30 uL TBE, 6 uL loading dye, and 4 uL sample for C1-C2 and C3-C2
    2. Mix 15 uL TBE, 3 uL loading dye, and 2 uL ladder
    3. Add 20 uL to each lane (2 lanes for C1-C2 and C3-C2)
    4. Run the gel at 250V for 25 min
    5. Stain with 3 uL SYBR gold for 3 min
    6. Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube
    7. Shred the gel by centrifuging at 14000rpm for 1 min 30 sec
    8. Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking
    9. Centrifuge at 12000rpm for 1.5 min
    10. Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm
  2. Ethanol Precipitation
    1. Move flow through to a 2 mL tube
    2. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue
    3. Incubate at -80C for 4 hours
    4. Pellet by centrifuging for 25 min at 12000rpm at 4C
    5. Remove supernatant and add 750 uL chilled 70% EtOH
    6. Centrifuge for 12 min at 12000 rpm and 4 C
    7. Remove supernatant and air dry
    8. Resuspend in 30 uL nfH2O and measure in nanodrop

    Nanodrop Results

    Positive Control Amplicon Test

    1. Latch and Padlock Hybridization
      1. Mix together samples according to table; use 100 uL LPH buffer with 100 nM Padlock0001-68
      2. Incubate for 1 hr at 37C
      3. Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
      4. Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
      5. Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
        1. For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)
      6. Incubate for 30 min at 37C
      7. Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
      8. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
      9. Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
    2. Circularization
      1. Prepare 20 uL KLN mix
      2. Prepare 4.1x master mix of Phusion mix
      3. Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
      4. Add 2 uL KLN mix to 20 uL of reaction for sample 1
      5. Incubate at 55C overnight (XX hours)
      6. Continued tomorrow
      Sample No Sample Name LatchX2 LatchX3 PCAmp1 PCAmp3 Expected Product
      1 Amp1 Test X   X   Amp1
      2 Amp3 Test   X   X Amp3
      3 LatchX2 Specificity X   X X Amp1
      4 LatchX3 Specificity   X X X Amp3
      5 No Template X X     None
      6 No Latch     X X None
      7 No Padlock X X X X None