Daniel:Notebook/ComboLock/2016-6-23: Difference between revisions
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==Protocol== | ==Protocol== | ||
===Prep=== | |||
'''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="110" height="42" | Reagent | |||
| width="65" | Stock | |||
| width="65" | Final | |||
| width="65" | Dilution | |||
| width="82" | Amt in 10 mL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | PBS | |||
| align="center" valign="bottom" | 10X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" valign="bottom" | 1 mL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Tween 20 | |||
| align="center" align="center" valign="bottom" | 100% | |||
| align="center" align="center" valign="bottom" | 0.10% | |||
| align="center" align="center" valign="bottom" | 1000 | |||
| align="center" valign="bottom" | 10 uL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Rnasin | |||
| align="center" valign="bottom" | 40000 U/mL | |||
| align="center" valign="bottom" | 4 U/mL | |||
| align="center" align="center" valign="bottom" | 1000 | |||
| align="center" valign="bottom" | 10 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | nf H2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | 9 mL | |||
|} | |||
'''Stringent 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="110" height="42" | Reagent | |||
| width="65" | Stock | |||
| width="65" | Final | |||
| width="65" | Dilution | |||
| width="74" | Amt in 1 mL (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | PBS | |||
| align="center" valign="bottom" | 10X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" valign="bottom" | 100 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | SSC | |||
| align="center" valign="bottom" | 20X | |||
| align="center" valign="bottom" | 4X | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" valign="bottom" | 200 uL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Rnasin | |||
| align="center" valign="bottom" | 40000 U/mL | |||
| align="center" valign="bottom" | 40 U/mL | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" valign="bottom" | 10 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | nf H2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | 690 uL | |||
|} | |||
'''LPH 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="110" height="42" | Reagent | |||
| width="65" | Stock | |||
| width="65" | Final | |||
| width="65" | Dilution | |||
| width="74" | Amt in 100 uL (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Oligos | |||
| align="center" valign="bottom" | 10 uM | |||
| align="center" valign="bottom" | 100 nM | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" valign="bottom" | 1 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | SSC | |||
| align="center" valign="bottom" | 20X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" valign="bottom" | 20 uL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Rnasin | |||
| align="center" valign="bottom" | 40000 U/mL | |||
| align="center" valign="bottom" | 40 U/mL | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" valign="bottom" | 1 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | PBS | |||
| align="center" valign="bottom" | 10X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" valign="bottom" | 10 uL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | nf H2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | 68 uL | |||
|} | |||
===Experiment=== | |||
'''Sample Matrix''' | |||
{| 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="85" height="30" | Sample | |||
| width="53" | VIM-C1 (1 uL) | |||
| width="52" | VIM-C2 (1 uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 1 | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | X | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 2 | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
|} | |||
<ol> | <ol> | ||
Line 12: | Line 161: | ||
<li>Split cells into 2 samples (1 mL methanol each)</li> | <li>Split cells into 2 samples (1 mL methanol each)</li> | ||
<li>Pellet cells via centrifugation at 600g for 3 min</li> | <li>Pellet cells via centrifugation at 600g for 3 min</li> | ||
<li>Combine 1.5 uL of each C1 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>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> | ||
<li>Add probes to | <li>Add probes to CPH buffer for final concentration of 100 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 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> | ||
<li>Incubate cells for 20 min at 40C in stringent wash buffer</li> | <li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li> | ||
</ol> | </ol> | ||
<li>Latch and Padlock Hybridization</li> | <li>Latch and Padlock Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Pellet at 600xg for 3 min</li> | <li>Pellet at 600xg for 3 min and remove supernatant</li> | ||
<li> | <li>Resuspend pellet in 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)</li> | ||
<li>Wash twice</li> | <li> Add 1 uL of each insert/backbone (final conc. 100 nM each)</li> | ||
<ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol></ol> | |||
'''Sample Matrix''' | |||
{| 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="65" height="30" | Batch | |||
| width="65" | Cell Sample | |||
| width="65" | Latch0001 (1 uL) | |||
| width="65" | Latch0002 (1 uL) | |||
| width="89" | Padlock0001-48 (1 uL) | |||
| width="87" | Padlock0001-68 (1 uL) | |||
| width="91" | Padlock0001-88 (1 uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Batch 1 | |||
| align="center" | S1 | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Batch 2 | |||
| align="center" | S1 | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" align="center" | | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Batch 3 | |||
| align="center" | S1 | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" | X | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Batch 4 | |||
| align="center" | S1 | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Batch 5 | |||
| align="center" | S1 | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" align="center" | | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Batch 6 | |||
| align="center" | S1 | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" | X | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Batch 7 | |||
| align="center" | S2 | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Batch 8 | |||
| align="center" | S2 | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
|} | |||
<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 with 20 uL 1x Amp Ligase buffer</li></ol> | |||
<li>Circularization</li> | <li>Circularization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Prepare KLN | <li>Prepare 20 uL KLN mix</li> | ||
<li>Add 2 uL KLN mix to 20 uL of reaction</li> | <li>Add 2 uL KLN mix to 20 uL of reaction</li> | ||
<li>Incubate at 55C | <li>Incubate at 55C overnight (XX hours) </li> | ||
<li> | <li>Continued [[Daniel:Notebook/ComboLock/2016-6-24|tomorrow]]</li> | ||
</ol> | </ol> | ||
===Buffers=== | |||
<ol type="A"> | <ol type="A"> | ||
<li> | <li>C-Probe Hybridization (CPH) Buffer</li> | ||
<li> | <ol type="a"> | ||
<li> | <li>100 nM insert/backbone oligos</li> | ||
<li> | <li>1X SSC</li> | ||
<li> | <li>40 U/mL RNasin</li> | ||
<li>PBS</li> | |||
</ol> | </ol> | ||
<li> | <li>Wash Buffer</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> | <li>PBS</li> | ||
<li> | <li>0.1% Tween</li> | ||
<li> | <li>4 U/mL RNasin</li> | ||
</ol> | </ol> | ||
<li>Latch-Padlock Hybridization (LPH) Buffer</li> | |||
<ol type="a"> | |||
<li>100 nM insert/backbone oligos</li> | |||
<li>1X SSC</li> | |||
<li>40 U/mL RNasin</li> | |||
<li>PBS</li> | |||
</ol> | </ol> | ||
<li>KLN Mix 20 uL total | |||
<ol type="a"> | |||
<li> 4 uL HemoKlentaq (20% v/v)</li> | |||
<li> 2 μL Ampligase (0.5 U/uL)</li> | |||
<li> 2 uL 1 mM dNTP mix (100 μM ea)</li> | |||
<ol type="i"><li>Add 10 uL 10mM dNTP mix to 90 uL nfH2O</li></ol> | |||
<li> 2 uL 1x Ampligase Buffer 1x</li> | |||
<li> 10 uL nfH2O</li> | |||
</ol> | </ol> | ||
[[Category:ComboLock]] [[Category:20160623]] |
Latest revision as of 22:30, 6 July 2016
Stage 2 Test[edit]
The purpose of this experiment is to test the latch and padlock binding, termed stage 2 (at the moment). This is based on the success of the VIM binding for both C-Probes and antibodies.
Protocol[edit]
Prep[edit]
Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 10 mL |
PBS | 10X | 1X | 10 | 1 mL |
Tween 20 | 100% | 0.10% | 1000 | 10 uL |
Rnasin | 40000 U/mL | 4 U/mL | 1000 | 10 uL |
nf H2O | NA | NA | NA | 9 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 | 100 | 10 uL |
nf H2O | NA | NA | NA | 690 uL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 100 uL (uL) |
Oligos | 10 uM | 100 nM | 100 | 1 uL |
SSC | 20X | 1X | 5 | 20 uL |
Rnasin | 40000 U/mL | 40 U/mL | 100 | 1 uL |
PBS | 10X | 1X | 10 | 10 uL |
nf H2O | NA | NA | NA | 68 uL |
Experiment[edit]
Sample Matrix
Sample | VIM-C1 (1 uL) | VIM-C2 (1 uL) |
Sample 1 | X | X |
Sample 2 |
- C-Probe Hybridization
- Split cells into 2 samples (1 mL methanol each)
- Pellet cells via centrifugation at 600g for 3 min
- Combine 1.5 uL of each C1 probe into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to CPH buffer for final concentration of 100 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)
- Add 1 uL of each insert/backbone (final conc. 100 nM each) Sample Matrix
- Incubate for 30 min at 37C following the sample matrix
- Wash twice with 200 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 20 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight (XX hours)
- Continued tomorrow
- C-Probe Hybridization (CPH) Buffer
- 100 nM insert/backbone oligos
- 1X SSC
- 40 U/mL RNasin
- PBS
- Wash Buffer
- PBS
- 0.1% Tween
- 4 U/mL RNasin
- Latch-Padlock Hybridization (LPH) Buffer
- 100 nM insert/backbone oligos
- 1X SSC
- 40 U/mL RNasin
- PBS
- KLN Mix 20 uL total
- 4 uL HemoKlentaq (20% v/v)
- 2 μL Ampligase (0.5 U/uL)
- 2 uL 1 mM dNTP mix (100 μM ea)
- Add 10 uL 10mM dNTP mix to 90 uL nfH2O
- 2 uL 1x Ampligase Buffer 1x
- 10 uL nfH2O
Batch | Cell Sample | Latch0001 (1 uL) | Latch0002 (1 uL) | Padlock0001-48 (1 uL) | Padlock0001-68 (1 uL) | Padlock0001-88 (1 uL) |
Batch 1 | S1 | X | X | |||
Batch 2 | S1 | X | X | |||
Batch 3 | S1 | X | X | |||
Batch 4 | S1 | X | X | |||
Batch 5 | S1 | X | X | |||
Batch 6 | S1 | X | X | |||
Batch 7 | S2 | X | X | |||
Batch 8 | S2 | X | X |