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

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<ol type="I">
<ol type="I">
<li>Ribonucleoside Vanadyl Complex-to minimize freeze thaws, thaw at 65C for 10 min; store in 1 mL aliquots</li>
<li>Resuspend following primers to 100 uM (pmol/uL)</li>
<ol type="i">
<li>'''C Probes (40 uL to 100 uM)''': CAV1-C1/C2,GFAP-C1/C2,SOD1-C1/C2,VIM-C1/C2</li>
<li>'''Primers (uL to 100 uM)''': P2 (295), P4_RC (295), P6 (316), P12-RC (323) </li>
</ol>
<li>Wash Buffer</li>
<li>Wash Buffer</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center"
| width="101" height="30" | Reagent
| width="85" | Stock Conc.
| width="71" | Final Conc.
| width="74" | Amt. Added
|- style="font-size:12pt"
| height="15"  valign="bottom" | PBS
| align="center" valign="bottom" | 10X
| align="center" valign="bottom" | 1X
| align="center" valign="bottom" | 5 mL
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Tween 20
| align="center" align="center" valign="bottom" | 100%
| align="center" align="center" valign="bottom" | 0.10%
| align="center" valign="bottom" | 100 uL
|- style="font-size:12pt"
| height="15"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 4 U/mL
| align="center" valign="bottom" | 5 uL
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Water
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 44.9 mL
|- style="font-size:12pt"
| height="15"  valign="bottom" | Total Volume
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" valign="bottom" | &nbsp;
|style="font-weight:bold" align="center" valign="bottom" | 50 mL
|}
<li>Hybridization Buffer-Make 10 mL at a time and store in 1 mL aliquots</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center"
| width="143" height="30" | Reagent
| width="101" | Stock Conc.
| width="86" | Final Conc.
| width="108" | Amt. Added
| width="65" | Amt Added
|- style="font-size:12pt"
| height="15"  valign="bottom" | SSC
| align="center" valign="bottom" | 20X
| align="center" valign="bottom" | 1X
| align="center" valign="bottom" |  50 uL
| align="center" valign="bottom" | 500 uL
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Tween 20
| align="center" align="center" valign="bottom" | 100%
| align="center" align="center" valign="bottom" | 0.10%
| align="center" valign="bottom" | 1 uL
| align="center" valign="bottom" | 10 uL
|- style="font-size:12pt"
| height="15"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" valign="bottom" | 1 uL
| align="center" valign="bottom" | 10 uL
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Riboside Vanadyl Complex
| align="center" valign="bottom" | 200 mM
| align="center" valign="bottom" | 20 mM
| align="center" valign="bottom" | 100 uL
| align="center" valign="bottom" | 1 mL
|- style="font-size:12pt"
| height="15"  valign="bottom" | Poly Vinylsulfonic Acid
| align="center" align="center" valign="bottom" | 25%
| align="center" align="center" valign="bottom" | 2.50%
| align="center" valign="bottom" | 100 uL
| align="center" valign="bottom" | 1 mL
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Salmon Sperm
| align="center" valign="bottom" | 10 mg/mL
| align="center" valign="bottom" | 100 ug/mL
| align="center" valign="bottom" | 10 uL
| align="center" valign="bottom" | 100 uL
|- style="font-size:12pt"
| height="15"  valign="bottom" | Probes
| align="center" valign="bottom" | 100 uM
| align="center" valign="bottom" | 100 nM
| align="center" valign="bottom" | 1 uL/probe
| align="center" valign="bottom" | ***
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Water
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 740 uL
| align="center" valign="bottom" | 7.4 mL
|- style="font-size:12pt"
| height="15"  valign="bottom" | Total
| align="center"  valign="bottom" | &nbsp;
| align="center"  valign="bottom" | &nbsp;
|style="font-weight:bold" align="center" valign="bottom" | 1 mL
|style="font-weight:bold" align="center" valign="bottom" | 10 mL
|}


===Probe Hybridization===
===Probe Hybridization===
Line 17: Line 134:


<ol>
<ol>
<li>Fix cells</li>
<li>Fix cells (CERC)</li>
<ol type="A">
<ol type="A">
<li>Start with confluent U87MG cells</li>
<li>Start with confluent U87MG cells</li>
Line 27: Line 144:
<li>Fix cells in 5 mL EMEM with 1.6% paraformaldehyde for 10 min at RT</li>
<li>Fix cells in 5 mL EMEM with 1.6% paraformaldehyde for 10 min at RT</li>
<li>Pellet cells by centrifuging at 600xg for 3 min</li>
<li>Pellet cells by centrifuging at 600xg for 3 min</li>
<li>Wash cells with wash buffer</li>
<li>Wash cells with 5 mL wash buffer</li>
<li>Pellet cells and permeabilize with ice-cold methanol for 10 min on ice</li>
<ol type="a">
Once in methanol cells may be stored long term
<li>add 0.5 uL RNasin (40 u/uL) to 5 mL wash buffer prime</li>
</ol>
</ol>
<li>Hybridization</li>
<li>Pellet cells and add 5 mL ice-cold methanol to permeabilize; incubate 10 min on ice</li>
<ol type="A">
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Add probes to cells for final concentration of 100 nM</li>
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li>
<li>Wash three times with wash buffer and pelleting at 600xg for 3 min</li>
<li>Incubate cells for 20 min at 40C in stringent wash buffer</li>
</ol>
</ol>
</ol>
</ol>
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<ol type="A">
<ol type="A">
<li> '''Wash Buffer''': 1X PBS, 0.1% Tween-20, 4 U/mL RNasin</li>
<li>'''Wash Buffer''': 1X PBS, 0.1% Tween-20, 4 U/mL RNasin</li>
<li>'''Hybridization Buffer''': 1X SSC, 20mM RVC, 40 U/mL RNasin, 0.1% Tween 20, 100 ug/mL salmon sperm DNA</li>
</ol>
</ol>

Latest revision as of 20:01, 23 May 2016

C Probe Hybridization Test[edit]

Back to Calendar

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