Daniel:Notebook/ComboLock/2016-7-11: Difference between revisions

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>Djacobse
(Created page with "=Million Fold Test= Back to Calendar After conversations with Dr. Zhang and Andrew on Friday I'm going to try a SOD1 test using 5X and 10X amou...")
 
>Djacobse
 
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{| 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>
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center"
| width="110" height="42" | Reagent
| width="65" height="30" | Reagent
| width="65" | Stock
| width="105" | Stock
| width="65" | Final
| width="65" | Final
| width="65" | Dilution
| width="65" | Dilution
| width="74" | Amt in 100 uL (uL)
| width="65" | Amt in 1 mL (uL)


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Oligos
|style="font-weight:bold" height="15"  valign="bottom" | Oligos
| align="center" valign="bottom" | 10 uM
| align="center" valign="bottom" | 100 uM
| align="center" valign="bottom" | 100 nM
| align="center" valign="bottom" | 100 nM
| align="center" align="center" valign="bottom" | 100
| align="center" align="center" valign="bottom" | 1000
| align="center" valign="bottom" | 1 uL
| align="center" valign="bottom" | 100 uL


|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#D9D9D9;font-size:12pt"
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| align="center" valign="bottom" | 20X
| align="center" valign="bottom" | 20X
| align="center" valign="bottom" | 1X
| align="center" valign="bottom" | 1X
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 20
| align="center" valign="bottom" | 20 uL
| align="center" valign="bottom" | 50 uL


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Rnasin
|style="font-weight:bold" height="30"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" align="center" valign="bottom" | 100
| align="center" align="center" valign="bottom" | 1000
| align="center" valign="bottom" | 1 uL
| align="center" valign="bottom" | 1 uL


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| align="center" valign="bottom" | 1X
| align="center" valign="bottom" | 1X
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 10 uL
| align="center" valign="bottom" | 100 uL


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | nf H2O
|style="font-weight:bold" height="30"  valign="bottom" | nf H2O
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 68 uL
| align="center" valign="bottom" | 850 uL


|}
|}
'''KLN Mix'''


{| 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>
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center"
| width="99" height="31" | Reagent
| width="180" height="31" | Reagent
| width="65" | Stock
| width="65" | Stock
| width="65" | uL added
| width="65" | uL added
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|}
|}
'''Wash Buffer'''
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center"
| width="105" height="30" | Reagent
| width="105" | Stock Conc.
| width="65" | Final Conc.
| width="65" | 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" | 50 uL
|- style="font-size:12pt"
| height="30"  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="30"  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
|}
Store in 5 mL aliquots


==Experiment==
==Experiment==
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<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Combine 17 uL of each C probe into a 0.2 mL tube</li>
<li>Combine 17 uL of each C probe into a 0.2 mL tube</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Add probes to CPH buffer according to sample matrix</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>
|- style="background-color:#CCC0DA;font-size:12pt" align="center" valign="bottom"
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center"
| width="104" height="30" | Sample
| width="104" height="45" | Sample
| width="65" | Cell Type
| width="65" | Cell Type
| width="65" | uL 100 uM C Probe
| width="65" | uL 100 uM C Probe
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| align="center" | 1
| align="center" | 1
  | 100 nM
  | 100 nM
| align="center" | 0.05
| align="center" | 0.01
| align="center" | 7.5
| align="center" | 6.8
| align="center" | 2
| align="center" | 2


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| align="center" | 5
| align="center" | 5
  | 500 nM
  | 500 nM
| align="center" | 0.25
| align="center" | 0.05
| align="center" | 8.2
| align="center" | 7.5
| align="center" | 10
| align="center" | 10


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| align="center" | 10
| align="center" | 10
  | 1 uM
  | 1 uM
| align="center" | 0.5
| align="center" | 0.1
| align="center" | 8.5
| align="center" | 7.8
| align="center" | 20
| align="center" | 20


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| align="center" | 1
| align="center" | 1
  | 100 nM
  | 100 nM
| align="center" | 0.05
| align="center" | 0.01
| align="center" | 7.5
| align="center" | 6.8
| align="center" | 2
| align="center" | 2


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| align="center" | 5
| align="center" | 5
  | 500 nM
  | 500 nM
| align="center" | 0.25
| align="center" | 0.05
| align="center" | 8.2
| align="center" | 7.5
| align="center" | 10
| align="center" | 10


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| align="center" | 10
| align="center" | 10
  | 1 uM
  | 1 uM
| align="center" | 0.5
| align="center" | 0.1
| align="center" | 8.5
| align="center" | 7.8
| align="center" | 20
| align="center" | 20


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| align="center" | 1
| align="center" | 1
  | 100 nM
  | 100 nM
| align="center" | 0.05
| align="center" | 0.01
  | NA
  | NA
| align="center" | 2
| align="center" | 2


|}
|}
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Add probes to CPH buffer for final concentration of 500 or 1000 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)</li>
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li>
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li>
<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li>
<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li>
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<ol type="A">
<ol type="A">
<li>Pellet at 600xg for 3 min and remove supernatant</li>
<li>Pellet at 600xg for 3 min and remove supernatant</li>
<li>Resuspend pellet in 100 uL LPH buffer; use Latch0001 and Padlock0001-68 </li>
<li>Resuspend pellet in 100 uL LPH buffer with 200 nM Padlock0001-68 </li>
<li>Incubate for 30 min at 37C following the sample matrix</li>
<li>Wash twice with 200 uL wash buffer</li>
<ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol>
<li>Resuspend pellet in 100 uL LPH buffer with 1 uM Latch0001</li>
<li>Incubate for 30 min at 37C following the sample matrix</li>
<li>Incubate for 30 min at 37C following the sample matrix</li>
<li>Wash twice with 200 uL wash buffer</li>
<li>Wash twice with 200 uL wash buffer</li>
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<li>Continued [[Daniel:Notebook/ComboLock/2016-7-12|tomorrow]]</li>
<li>Continued [[Daniel:Notebook/ComboLock/2016-7-12|tomorrow]]</li>
</ol>
</ol>
[[Category:ComboLock]] [[Category:20160711]]

Latest revision as of 17:40, 12 July 2016

Million Fold Test[edit]

Back to Calendar

After conversations with Dr. Zhang and Andrew on Friday I'm going to try a SOD1 test using 5X and 10X amounts from the original protocol.

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 100 10 uL
nf H2O NA NA NA 690 uL

LPH Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
Oligos 100 uM 100 nM 1000 100 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

Wash Buffer

Reagent Stock Conc. Final Conc. Amt. Added
PBS 10X 1X 5 mL
Tween 20 100% 0.10% 50 uL
Rnasin 40000 U/mL 4 U/mL 5 uL
Water NA NA 44.9 mL
Total Volume     50 mL

Store in 5 mL aliquots

Experiment[edit]

  1. C-Probe Hybridization
    1. Measure cell concentrations in cell counter
      1. Human - cells/mL; Mouse - cells/mL
    2. Add cells to a new tube such that there are 100000 cells present
      1. Human- uL; Mouse- uL
    3. Pellet cells via centrifugation at 600g for 3 min
    4. Combine 17 uL of each C probe into a 0.2 mL tube
    5. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    6. Add probes to CPH buffer according to sample matrix
    7. Sample Cell Type uL 100 uM C Probe Final C Probe Concentration Final C Probe Amount (nmol) Estimated Fold (log10) uL Mix to add
      Hs-1 U87MG 1 100 nM 0.01 6.8 2
      Hs-2 U87MG 5 500 nM 0.05 7.5 10
      Hs-3 U87MG 10 1 uM 0.1 7.8 20
      Hs-4 U87MG 0 0 0 NA 0
      mm-1 3T3 1 100 nM 0.01 6.8 2
      mm-2 3T3 5 500 nM 0.05 7.5 10
      mm-3 3T3 10 1 uM 0.1 7.8 20
      mm-4 3T3 0 0 0 NA 0
      NTC NA 1 100 nM 0.01 NA 2
    8. Incubate probes at 40C for 1 hour with vigorous agitation
    9. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    10. 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 200 nM Padlock0001-68
    3. Incubate for 30 min at 37C following the sample matrix
    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 1 uM Latch0001
    6. Incubate for 30 min at 37C following the sample matrix
    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