Daniel:Notebook/ComboLock/2016-6-23

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Revision as of 22:58, 22 June 2016 by >Djacobse (→‎Protocol)
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Stage 2 Test

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The purpose of this experiment is to test the latch and padlock binding, termed stage 2 (at the moment). This is based on the success of the VIM binding for both C-Probes and antibodies.

Protocol

Sample Matrix

Sample VIM-C1 (1 uL) VIM-C2 (1 uL)
Sample 1 X X
Sample 2    
  1. C-Probe Hybridization
    1. Split cells into 2 samples (1 mL methanol each)
    2. Pellet cells via centrifugation at 600g for 3 min
    3. Put 1 uL C1 Probe and 1 uL C2 probe into a 0.2 mL tube
    4. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    5. Add probes to CPH buffer for final concentration of 100 nM (Add 1 uL probe mixture to 0.5mL hybridization buffer)
    6. Incubate probes at 40C for 1 hour with vigorous agitation
    7. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    8. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
  2. Latch and Padlock Hybridization
    1. Pellet at 600xg for 3 min and remove supernatant
    2. Resuspend pellet in 50 uL LPH buffer with 100 nM insert/backbone oligos for 30 min at 37C
    3. Wash twice with 200 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
  3. Circularization
    1. Prepare KLN mix with:
      1. 20% v/v HemoKlentaq
      2. 0.5 U/μL Ampligase
      3. 100 μM of dNTP mix
      4. 1x Ampligase Buffer
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight for 4-20 hours
    4. Continued tomorrow

    Buffers

    1. C-Probe Hybridization (CPH) Buffer
      1. 100 nM insert/backbone oligos
      2. 1X SSC
      3. 40 U/mL RNasin
      4. PBS
    2. Wash Buffer
      1. PBS
      2. 0.1% Tween
      3. 4 U/mL RNasin
    3. Latch-Padlock Hybridization (LPH) Buffer