Daniel:Notebook/ComboLock/2016-5-23: Difference between revisions

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<li>Ribonucleoside Vanadyl Complex-to minimize freeze thaws, thaw at 65C for 10 min; store in 1 mL aliquots</li>
<li>Wash Buffer</li>
<li>Wash Buffer</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>

Revision as of 18:45, 23 May 2016

C Probe Hybridization Test

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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

Buffer and Reagent Prep

  1. Ribonucleoside Vanadyl Complex-to minimize freeze thaws, thaw at 65C for 10 min; store in 1 mL aliquots
  2. Wash Buffer
  3. 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
  4. Hybridization Buffer-Make 10 mL at a time and store in 1 mL aliquots
  5. 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

    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
    2. Hybridization (Lab)
      1. Pellet cells via centrifugation at 600g for 3 min
      2. Heat C probes to 90C for 5 min; Chill probes on ice to quench
      3. Add probes to cells for final concentration of 100 nM
      4. Incubate probes at 40C for 1 hour with vigorous agitation
      5. Wash three times with wash buffer and pelleting at 600xg for 3 min
      6. Incubate cells for 20 min at 40C in stringent wash buffer

    Buffers

    1. Wash Buffer: 1X PBS, 0.1% Tween-20, 4 U/mL RNasin