Daniel:Notebook/ComboLock/2016-10-5: Difference between revisions
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[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
===Buffers=== | |||
'''Wash/Binding 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="65" height="30" | Reagent | |||
| width="65" | Stock | |||
| width="65" | Final | |||
| width="65" | Dilution | |||
| width="65" | Amt in 10 mL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | NaCl | |||
| align="center" valign="bottom" | 1.5 M | |||
| align="center" valign="bottom" | 0.5 M | |||
| align="center" align="center" valign="bottom" | 3 | |||
| align="center" valign="bottom" | 3.33 mL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Tris-HCl | |||
| align="center" valign="bottom" | 500 mM | |||
| align="center" valign="bottom" | 20 mM | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" valign="bottom" | 400 uL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | EDTA | |||
| align="center" valign="bottom" | 0.5M | |||
| align="center" valign="bottom" | 1 mM | |||
| align="center" align="center" valign="bottom" | 500 | |||
| align="center" valign="bottom" | 20 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" | 6.25 mL | |||
|} | |||
==Protocol== | ==Protocol== | ||
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<ol> | <ol> | ||
<li>Template-Bead Binding</li> | <li>Template-Bead Binding</li> | ||
<ol type="A"> | |||
<li>Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer </li> | <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>Apply magnet for 30 sec and remove supernatant</li> | ||
Line 44: | Line 87: | ||
<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 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</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> | </ol> | ||
<li>Latch and Padlock Hybridization</li> | <li>Latch and Padlock Hybridization</li> |
Revision as of 19:56, 5 October 2016
C Probe Positive Control Test
Buffers
Wash/Binding Buffer
Reagent | Stock | Final | Dilution | Amt in 10 mL |
NaCl | 1.5 M | 0.5 M | 3 | 3.33 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 400 uL |
EDTA | 0.5M | 1 mM | 500 | 20 uL |
nf H2O | NA | NA | NA | 6.25 mL |
Protocol
C Probes-VIM (C1+C2) | C Probes-SOD1 (C1+C2) | Template | |
Sample 1 | X | X | X |
Sample 2 | X | X | |
Sample 3 | X |
- 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 and 3 uL CPH 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 30 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight; continued tomorrow