Daniel:Notebook/ComboLock/2016-6-23

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Revision as of 22:46, 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 hybridization buffer for final concentration of 100 nM (0.5 uL/probe/sample using 0.5mL hybridization buffer)
    6. Incubate probes at 40C for 1 hour with vigorous agitation
    7. Wash three times with wash buffer and pelleting at 600xg for 3 min
    8. Incubate cells for 20 min at 40C in stringent wash buffer
  2. Latch and Padlock Hybridization
    1. Pellet at 600xg for 3 min
    2. Incubate with 100 nM insert/backbone oligos for 30 min at 37C
    3. Wash twice
      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 for 4-20 hours
    4. Heat inactivate enzyme by incubating for 2 minutes at 94C
  4. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing exonuclease I (20 units/μL) and exonuclease III (200 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  5. qPCR
    1. Set up reaction according to table
    2. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x20
      7. 72C 2 min
      8. 16C hold