Daniel:Notebook/ComboLock/2017-3-13: Difference between revisions
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<li>Repeat wash step above</li> | <li>Repeat wash step above</li> | ||
<li>Repeat wash step above</li> | <li>Repeat wash step above</li> | ||
</ol> | </ol></ol> | ||
</ol> | |||
==Protocol-Part 2-LB hybridization & ligation== | |||
<ol start="3"> | |||
<li>Bolt and Latch Hybridization/Ligation</li> | |||
<ol type="A"> | |||
<li>Set up the following master mix</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | |||
| width="200" height="40" | Reagent | |||
| width="85" | Stock Conc | |||
| width="85" | Final Conc./Amt | |||
| width="85" | uL added | |||
| width="85" | Master Mix (8.2X) | |||
|- style="font-size:12pt" | |||
| height="30" valign="bottom" | T4 Ligase Reaction Buffer | |||
| align="center" | 5X | |||
| align="center" | 1X | |||
| align="center" align="center" | 4 | |||
| align="center" align="center" valign="bottom" | 32.8 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Latch Oligo | |||
| align="center" | 10 uM | |||
| align="center" | 10 pmol | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" valign="bottom" | 8.2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Lock Oligo | |||
| align="center" | 10 uM | |||
| align="center" | 10 pmol | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" valign="bottom" | 8.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" | NA | |||
| align="center" | NA | |||
| align="center" align="center" | 13 | |||
| align="center" align="center" valign="bottom" | 106.6 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Total | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
|style="font-weight:bold" align="center" align="center" | 19 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 155.8 | |||
|} | |||
<li>Add 19 uL probe mix to each sample</li> | |||
<li>Incubate at 55C for 10 minutes</li> | |||
<li>Incubate at 25C for 10 minutes</li> | |||
<li>Lower the temperature to 20C; wait 30 seconds then take off incubator</li> | |||
<li>Add 1 uL T4 Ligase to each reaction</li> | |||
<li>Incubate at RT for 15 min</li> | |||
<li>Heat kill enzyme by incubating at 65C for 10 minutes</li> | |||
</ol></ol> |
Revision as of 15:58, 13 March 2017
Bolt Test (Started Wednesday March 8)
Protocol
Sample Matrix
Sample | Condition |
Sample 5 (AB) | No Ligase |
Sample 6 (AB) | Normal |
- 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 following into a 0.2 mL tube
- 4 uL each PCCA-03 and PCCB-03 (10 uM each)
- 12 uL wash buffer
- 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
- Repeat wash step above
Protocol-Part 2-LB hybridization & ligation
- Bolt and Latch Hybridization/Ligation
- Set up the following master mix
- Add 19 uL probe mix to each sample
- Incubate at 55C for 10 minutes
- Incubate at 25C for 10 minutes
- Lower the temperature to 20C; wait 30 seconds then take off incubator
- Add 1 uL T4 Ligase to each reaction
- Incubate at RT for 15 min
- Heat kill enzyme by incubating at 65C for 10 minutes
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (8.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 32.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 8.2 |
Lock Oligo | 10 uM | 10 pmol | 1 | 8.2 |
nfH2O | NA | NA | 13 | 106.6 |
Total | 19 | 155.8 |