Daniel:Notebook/ComboLock/2016-7-5

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Sequential Hybridization Test[edit]

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Today I'm going to try testing the latch and padlock hybridizations sequentially. This is because of the unusual results of the C probe tests from and June 30 and July 2.

Protocol[edit]

Buffer Prep[edit]

C Probe Hyridization (CPH) Buffer

Reagent Stock Conc. Final Conc. Amt. Added Amt Added
SSC 20X 1X 50 uL 500 uL
Tween 20 100% 0.10% 1 uL 10 uL
Rnasin 40000 U/mL 40 U/mL 1 uL 10 uL
Riboside Vanadyl Complex 200 mM 20 mM 100 uL 1 mL
Poly Vinylsulfonic Acid 25% 2.50% 100 uL 1 mL
Salmon Sperm 10 mg/mL 100 ug/mL 10 uL 100 uL
Probes 100 uM 100 nM 1 uL/probe ***
Water NA NA 740 uL 7.4 mL
Total     1 mL 10 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 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

Experiment[edit]

  1. C-Probe Hybridization
    1. Transfer cells to a new tube such that there are 100000 cells present
      1. Human-1000 uL; Mouse-85 uL
      2. Human cells from July 2; 1.17E5 cells/mL
    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 Latch0001
    3. Incubate for 30 min at 37C
    4. Wash twice with 200 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
    5. Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0001-68
    6. Incubate for 30 min at 37C
    7. Wash twice with 200 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
    8. 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

    SOD1 Antibody Prep[edit]

    Since I have the VIM antibody and C Probes ready and only the SOD1 C Probe, it makes sense to have this antibody as well.

    Protocol[edit]

    1. Prep
      1. Resuspend SOD1-PA in 63 uL nfH2O (1 mM)
      2. Resuspend SOD1-PB in 47 uL nfH2O (1 mM)
    2. Antibody Activation
      1. Resuspend SOD1 with 100 uL PBS (final conc 1 mg/mL)
      2. Do this twice:
        1. Equilibrate a 40 kDa spin column 4 times with 100 uL PBS, (100 mM pH 7.3)
          1. Spin at 1000xg for 2 min
        2. Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube
        3. Add 2 uL 3.3 mM sulfo-SMCC in 100 mM phosphate buffer to sample
          1. Stock is 100 mM sulfo-SMCC in 100 mM phosphate buffer
          2. Mix 3.3 uL stock with 96 uL 100 mM phosphate buffer (dilute from 1 M stock)
        4. Incubate for 2 hr at 4C with 3 times intermittent mixing
        5. Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
        6. Transfer antibodies to new column and spin for 3 min at 1000xg
    3. Oligonucleotide Activation
      1. For VIM-PA and VIM-PB:
        1. Add 1.3 uL 100 uM oligonucleotide to tube
        2. Add 2.2 uL 40 mM DTT
        3. Incubate at 95C for 2 min followed by 1 hr at 37C
        4. Add 20 uL PBS with 20 mM EDTA
        5. Remove excess DTT using two consecutive Zeba 7 kDa columns equilibrated with 100 mM PBS (10X PBS)
          1. Spin at 1500xg for 1 minute for equilibrium; 2 min for sample
    4. Ab-Oligo Mixing
      1. For VIM-PA and VIM-PB:
        1. Mix SMCC-treated antibodies (22 uL) with DTT-treated oligonucleotides (35 uL)
          1. Amounts to 10X molar excess of oligo to Ab
          2. Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup
          3. Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS
          4. Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl