Daniel:Notebook/ComboLock/2016-10-7: Difference between revisions
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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=" | | width="120" height="39" | Reagent | ||
| width="65" | Stock | | width="65" | Stock | ||
| width="65" | uL added | | width="65" | uL added | ||
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<li>Latch and Padlock Hybridization</li> | <li>Latch and Padlock Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM | <li>Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0301 to each sample to a fresh 0.2 mL tube</li> | ||
<li>Heat at 65C for 5 minutes</li> | <li>Heat at 65C for 5 minutes</li> | ||
<li>Chill on ice for 3 minutes</li> | <li>Chill on ice for 3 minutes</li> |
Latest revision as of 16:40, 20 October 2016
Padlock0301 Test[edit]
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[edit]
Stock Dilutions[edit]
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[edit]
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[edit]
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[edit]
- 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 Padlock0301 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