Daniel:Notebook/ComboLock/2016-5-23

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Revision as of 20:01, 23 May 2016 by >Djacobse (→‎Protocol)
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C Probe Hybridization Test[edit]

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This will be the first test of the C Probe hybridization protocol for Combo-Lock. I have been growing U87MG cells for several days now and am ready to passage them. When I passage I'll take the remaining 90% for this test. Since I don't have the antibodies ready yet (I am waiting on some columns) I am just testing the C Probes.

Protocol[edit]

Buffer and Reagent Prep[edit]

  1. Ribonucleoside Vanadyl Complex-to minimize freeze thaws, thaw at 65C for 10 min; store in 1 mL aliquots
  2. Resuspend following primers to 100 uM (pmol/uL)
    1. C Probes (40 uL to 100 uM): CAV1-C1/C2,GFAP-C1/C2,SOD1-C1/C2,VIM-C1/C2
    2. Primers (uL to 100 uM): P2 (295), P4_RC (295), P6 (316), P12-RC (323)
  3. Wash Buffer
  4. Reagent Stock Conc. Final Conc. Amt. Added
    PBS 10X 1X 5 mL
    Tween 20 100% 0.10% 100 uL
    Rnasin 40000 U/mL 4 U/mL 5 uL
    Water NA NA 44.9 mL
    Total Volume     50 mL
  5. Hybridization Buffer-Make 10 mL at a time and store in 1 mL aliquots
  6. 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

    Probe Hybridization[edit]

    Base Protocol

    1. Fix cells (CERC)
      1. Start with confluent U87MG cells
      2. Remove media (EMEM) and wash with 1X PBS
      3. Trypsinize cells using 0.5 mL trypsin LE express; incubate 5 min at 37C
      4. Resuspend cells in by adding 4.5 mL media; use 0.5 mL to passage
      5. Pellet remainder of the cells in a 15 mL Falcon tube at 600g for 3 min
      6. Remove supernatant
      7. Fix cells in 5 mL EMEM with 1.6% paraformaldehyde for 10 min at RT
      8. Pellet cells by centrifuging at 600xg for 3 min
      9. Wash cells with 5 mL wash buffer
        1. add 0.5 uL RNasin (40 u/uL) to 5 mL wash buffer prime
      10. Pellet cells and add 5 mL ice-cold methanol to permeabilize; incubate 10 min on ice

    Buffers[edit]

    1. Wash Buffer: 1X PBS, 0.1% Tween-20, 4 U/mL RNasin
    2. Hybridization Buffer: 1X SSC, 20mM RVC, 40 U/mL RNasin, 0.1% Tween 20, 100 ug/mL salmon sperm DNA