Daniel:Notebook/ComboLock/2016-10-7: Difference between revisions
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==Protocol== | ==Protocol== | ||
===Stock Dilutions=== | |||
Create the following dilutions of PCC Template by taking 10 uL stock and diluting with 90 uL nfH2O (note:zero is normal 10 uM stock) | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="45" | Tube No | |||
| width="65" | Dilution | |||
| width="65" | Molecules Available | |||
| width="65" | Amount | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 0 | |||
| align="center" | 1 | |||
| align="center" | 1.00E+12 | |||
| 10 pmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" | |||
| align="center" height="15" | 1 | |||
| align="center" | 10 | |||
| align="center" | 1.00E+11 | |||
| 1 pmol | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 2 | |||
| align="center" | 100 | |||
| align="center" | 1.00E+10 | |||
| 100 fmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" | |||
| align="center" height="15" | 3 | |||
| align="center" | 1000 | |||
| align="center" | 1.00E+09 | |||
| 10 fmol | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 4 | |||
| align="center" | 10000 | |||
| align="center" | 1.00E+08 | |||
| 1 fmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" | |||
| align="center" height="15" | 5 | |||
| align="center" | 1.00E+05 | |||
| align="center" | 1.00E+07 | |||
| 100 amol | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 6 | |||
| align="center" | 100000 | |||
| align="center" | 1.00E+06 | |||
| 10 amol | |||
|} | |||
===Samples=== | |||
{| 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="99" height="45" | Sample | |||
| width="65" | C Probes-VIM (C1+C2) | |||
| width="65" | C Probes-SOD1 (C1+C2) | |||
| width="65" | Template | |||
| width="65" | Dilution | |||
| width="65" | Dilution Amount | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1 (ABC) | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" | 10 pmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2 | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | None | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 3 | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" valign="bottom" | 10 pmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 4 (ABC) | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | X | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" valign="bottom" | 100 fmol | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 5 (ABC) | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | X | |||
| align="center" align="center" valign="bottom" | 4 | |||
| align="center" valign="bottom" | 1 fmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 6 (ABC) | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | X | |||
| align="center" align="center" valign="bottom" | 6 | |||
| align="center" valign="bottom" | 10 amol | |||
|} | |||
===Buffers=== | |||
{| 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="82" height="39" | Reagent | |||
| width="65" | Stock | |||
| width="65" | uL added | |||
|- style="font-size:12pt" | |||
| height="30" valign="bottom" | Amp Ligase Buffer | |||
| align="center" valign="bottom" | 10X | |||
| align="center" align="center" valign="bottom" | 3 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | dNTPs | |||
| align="center" valign="bottom" | 100 uM | |||
| align="center" align="center" valign="bottom" | 3 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Hemo Klentaq | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 6 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase | |||
| align="center" valign="bottom" | 5 U/uL | |||
| align="center" align="center" valign="bottom" | 3 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 15 | |||
|} | |||
===Experiment Protocol=== | |||
<ol> | |||
<li>Template-Bead Binding</li> | |||
<ol type="A"> | |||
<li>Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer </li> | |||
<li>Apply magnet for 30 sec and remove supernatant</li> | |||
<li>Add 2 uL template oligo per sample to bead solution; incubate at RT for 5 min</li> | |||
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | |||
</ol> | |||
<li>C Probe Hybridization</li> | |||
<ol type="A"> | |||
<li>Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer 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 5 uL probe mixture to beads</li> | |||
<li>Incubate at 40C for 1 hour with agitation</li> | |||
<li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li> | |||
<li>Repeat wash step above</li> | |||
<li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li> | |||
</ol> | |||
<li>Latch and Padlock Hybridization</li> | |||
<ol type="A"> | |||
<li>Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube</li> | |||
<li>Heat at 65C for 5 minutes</li> | |||
<li>Chill on ice for 3 minutes</li> | |||
<li>Add sample to prepared strep beads; vortex to suspend</li> | |||
<li>Incubate for 30 min at 37C with intermittent mixing</li> | |||
<li>Apply magnet and remove supernatant</li> | |||
<li>Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant</li> | |||
<li>Repeat wash step above</li> | |||
<li>Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant</li> | |||
<li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol> | |||
<li>Circularization</li> | |||
<ol type="A"> | |||
<li>Prepare 10 uL KLN mix</li> | |||
<li>Add 2 uL KLN mix to 20 uL of reaction</li> | |||
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-10-8|tomorrow]] </li> | |||
</ol></ol> | |||
[[Category:ComboLock]] [[Category:20161007]] | [[Category:ComboLock]] [[Category:20161007]] |
Revision as of 19:07, 7 October 2016
Padlock0301 Test
This is a test of the padlock0301, which replaces the AmpF/LinkerR region of the padlock with a custom primer set. This is because my experiments on 9-29, 9-30, 10-3, and 10-4, which test the v1 and v2 padlock systems using AmpF and AmpR individually. AmpF has proved particularly dirty so I have designed v3 of the padlocks without it.
Protocol
Stock Dilutions
Create the following dilutions of PCC Template by taking 10 uL stock and diluting with 90 uL nfH2O (note:zero is normal 10 uM stock)
Tube No | Dilution | Molecules Available | Amount |
0 | 1 | 1.00E+12 | 10 pmol |
1 | 10 | 1.00E+11 | 1 pmol |
2 | 100 | 1.00E+10 | 100 fmol |
3 | 1000 | 1.00E+09 | 10 fmol |
4 | 10000 | 1.00E+08 | 1 fmol |
5 | 1.00E+05 | 1.00E+07 | 100 amol |
6 | 100000 | 1.00E+06 | 10 amol |
Samples
Sample | C Probes-VIM (C1+C2) | C Probes-SOD1 (C1+C2) | Template | Dilution | Dilution Amount |
Sample 1 (ABC) | X | X | X | 0 | 10 pmol |
Sample 2 | X | X | NA | None | |
Sample 3 | X | 0 | 10 pmol | ||
Sample 4 (ABC) | X | X | X | 2 | 100 fmol |
Sample 5 (ABC) | X | X | X | 4 | 1 fmol |
Sample 6 (ABC) | X | X | X | 6 | 10 amol |
Buffers
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 3 |
dNTPs | 100 uM | 3 |
Hemo Klentaq | NA | 6 |
Amp Ligase | 5 U/uL | 3 |
nfH2O | NA | 15 |
Experiment Protocol
- Template-Bead Binding
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL template oligo per sample to bead solution; incubate at RT for 5 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- C Probe Hybridization
- Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
- Latch and Padlock Hybridization
- Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube
- Heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C with intermittent mixing
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 10 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight; continued tomorrow