Daniel:Notebook/ComboLock/2016-10-5: Difference between revisions
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>Djacobse |
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Line 42: | Line 42: | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
| align="center" valign="bottom" | 6.25 mL | | align="center" valign="bottom" | 6.25 mL | ||
|} | |||
'''Low Salt 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" | 0.15 M | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 1 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" 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" 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" align="center" valign="bottom" | 8.58 mL | |||
|} | |||
'''KLN Mix''' | |||
{| 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="180" height="31" | Reagent | |||
| width="65" | Stock | |||
| width="65" | uL added | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase Buffer | |||
| align="center" valign="bottom" | 10X | |||
| align="center" align="center" valign="bottom" | 1 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | dNTPs | |||
| align="center" valign="bottom" | 100 uM | |||
| align="center" align="center" valign="bottom" | 1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Hemo Klentaq | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- 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" | 1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 5 | |||
|} | |} | ||
Line 79: | Line 153: | ||
<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> | ||
<li>Add 2 uL template oligo per sample to bead solution; incubate at RT for 5 min</li> | <li>Add 2 uL 10 uM 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> | <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | ||
</ol> | </ol> | ||
<li>C Probe Hybridization</li> | <li>C Probe Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Combine 1 uL of each C probe and 3 uL | <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>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> | ||
Line 106: | Line 180: | ||
<li>Circularization</li> | <li>Circularization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Prepare | <li>Prepare 10 uL KLN mix</li> | ||
<li>Add 2 uL KLN mix to 20 uL of reaction</li> | <li>Add 2 uL KLN mix to 20 uL of reaction</li> | ||
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-10-6|tomorrow]] </li> | <li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-10-6|tomorrow]] </li> | ||
</ol></ol> | </ol></ol> | ||
[[Category:ComboLock]] [[Category:20161005]] |
Latest revision as of 18:42, 7 October 2016
C Probe Positive Control Test[edit]
Buffers[edit]
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 |
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 |
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 1 |
dNTPs | 100 uM | 1 |
Hemo Klentaq | NA | 2 |
Amp Ligase | 5 U/uL | 1 |
nfH2O | NA | 5 |
Protocol[edit]
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 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
- 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