Daniel:Notebook/ComboLock/2016-12-13: Difference between revisions
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>Djacobse (Created page with "=Mirror Probe Test= Back to Calendar This is a continuation of the failed experiment from Friday with a...") |
>Djacobse |
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<li>Add 5 uL probe mixture to beads</li> | <li>Add 5 uL probe mixture to beads</li> | ||
<li>Incubate at 40C for 1 hour with agitation; '''start part 3 after incubation begins'''</li> | <li>Incubate at 40C for 1 hour with agitation; '''start part 3 after incubation begins'''</li> | ||
</ol> | |||
<li>Latch and Padlock Hybridization</li> | |||
<ol type="A"> | |||
<li>Add following to a fresh 0.2 mL tube</li> | |||
<ol type="a"> | |||
<li>12.2 uL Latch0001, 10 uM stock</li> | |||
<li>12.2 uL Padlock0201, 10 uM stock</li> | |||
<li>12.2 uL Amp10X buffer</li> | |||
<li>85.4 uL nfH2O</li></ol> | |||
<li>Add 20 uL probe mix to each sample; incubate 20C for 4 min</li> | |||
<li>Incubate at 95C for 5 min</li> | |||
<li>Incubate at 60C for 1.5 hours</li></ol> | |||
<li>Circularization</li> | |||
<ol type="A"> | |||
<li>Prepare 7X uL Phusion mix</li> | |||
<li>Add 20 uL Phusion mix to 20 uL of reaction</li> | |||
<li>Incubate at 60C for 2 hours</li></ol> | |||
</ol> | </ol> | ||
Continued [[Daniel:Notebook/ComboLock/2016-12-14|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20161213]] | |||
===Buffers=== | ===Buffers=== |
Revision as of 21:37, 13 December 2016
Mirror Probe Test
This is a continuation of the failed experiment from Friday with a corrected version of the template.
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probe |
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 10 uM 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; start part 3 after incubation begins
- Latch and Padlock Hybridization
- Add following to a fresh 0.2 mL tube
- 12.2 uL Latch0001, 10 uM stock
- 12.2 uL Padlock0201, 10 uM stock
- 12.2 uL Amp10X buffer
- 85.4 uL nfH2O
- Add 20 uL probe mix to each sample; incubate 20C for 4 min
- Incubate at 95C for 5 min
- Incubate at 60C for 1.5 hours
- Circularization
- Prepare 7X uL Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 60C for 2 hours
Continued tomorrow
Buffers
Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 20 mL |
NaCl | 1.5 M | 0.5 M | 3 | 6.66 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 800 uL |
EDTA | 0.5M | 1 mM | 500 | 40 uL |
nf H2O | NA | NA | NA | 12.5 mL |
Low Salt Buffer
Reagent | Stock | Final | Dilution | Amt in 10 mL |
NaCl | 1.5 M | 0.15 M | 10 | 1 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 400 uL |
EDTA | 0.5M | 1 mM | 500 | 20 uL |
nf H2O | NA | NA | NA | 8.58 mL |