Daniel:Notebook/ComboLock/2016-6-23

From ZhangLabWiki
Jump to navigation Jump to search

Stage 2 Test[edit]

Back to Calendar

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[edit]

Prep[edit]

Wash Buffer

Reagent Stock Final Dilution Amt in 10 mL
PBS 10X 1X 10 1 mL
Tween 20 100% 0.10% 1000 10 uL
Rnasin 40000 U/mL 4 U/mL 1000 10 uL
nf H2O NA NA NA 9 mL

Stringent Wash Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
PBS 10X 1X 10 100 uL
SSC 20X 4X 5 200 uL
Rnasin 40000 U/mL 40 U/mL 100 10 uL
nf H2O NA NA NA 690 uL

LPH Buffer

Reagent Stock Final Dilution Amt in 100 uL (uL)
Oligos 10 uM 100 nM 100 1 uL
SSC 20X 1X 5 20 uL
Rnasin 40000 U/mL 40 U/mL 100 1 uL
PBS 10X 1X 10 10 uL
nf H2O NA NA NA 68 uL

Experiment[edit]

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. Combine 1.5 uL of each C1 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 2 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 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)
    3. Add 1 uL of each insert/backbone (final conc. 100 nM each)
    4. Sample Matrix
      Batch Cell Sample Latch0001 (1 uL) Latch0002 (1 uL) Padlock0001-48 (1 uL) Padlock0001-68 (1 uL) Padlock0001-88 (1 uL)
      Batch 1 S1 X   X    
      Batch 2 S1 X     X  
      Batch 3 S1 X       X
      Batch 4 S1   X X    
      Batch 5 S1   X   X  
      Batch 6 S1   X     X
      Batch 7 S2 X   X    
      Batch 8 S2   X X    
    5. Incubate for 30 min at 37C following the sample matrix
    6. Wash twice with 200 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
    7. Resuspend with 20 uL 1x Amp Ligase buffer
  3. Circularization
    1. Prepare 20 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight (XX hours)
    4. Continued tomorrow

    Buffers[edit]

    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
      1. 100 nM insert/backbone oligos
      2. 1X SSC
      3. 40 U/mL RNasin
      4. PBS
    4. KLN Mix 20 uL total
      1. 4 uL HemoKlentaq (20% v/v)
      2. 2 μL Ampligase (0.5 U/uL)
      3. 2 uL 1 mM dNTP mix (100 μM ea)
        1. Add 10 uL 10mM dNTP mix to 90 uL nfH2O
      4. 2 uL 1x Ampligase Buffer 1x
      5. 10 uL nfH2O