Daniel:Notebook/ComboLock/2016-7-18

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C Probe Test 3-SOD1/VIM-Stringent Washes[edit]

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Protocol[edit]

Buffer Prep[edit]

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 1000 1 uL
nf H2O NA NA NA 700 uL

LPH Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
Oligos 100 uM 100 nM 1000 1 uL
SSC 20X 1X 20 50 uL
Rnasin 40000 U/mL 40 U/mL 1000 1 uL
PBS 10X 1X 10 100 uL
nf H2O NA NA NA 850 uL

KLN Mix

Reagent Stock uL added
Amp Ligase Buffer 10X 2
dNTPs 100 uM 2
Hemo Klentaq NA 4
Amp Ligase 5 U/uL 2
nfH2O NA 10

Resuspended SOD1 C probes in 40 uL nfH2O each (100 uM)

Experiment[edit]

  1. C-Probe Hybridization
    1. Add cells to a new tube such that there are 100000 cells present
      1. Human-500 uL; Mouse-140 uL
    2. Pellet cells via centrifugation at 600g for 3 min
    3. Combine 1.5 uL of each C 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.   C Probes-VIM (C1+C2) C Probes-SOD1 (C1+C2) U87MG 3T3 Cells
      Sample 1 X X X  
      Sample 2     X  
      Sample 3 X X   X
      Sample 4       X
      Sample 5 X X    
    7. Incubate probes at 40C for 1 hour with vigorous agitation
    8. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    9. 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 with 100 nM Padlock0001-68
    3. Incubate for 30 min at 37C
    4. Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
    5. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
    6. Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
    7. Incubate for 30 min at 37C
    8. Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
    9. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
    10. 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