Daniel:Notebook/ComboLock/2016-8-27: Difference between revisions
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>Djacobse (Created page with "=Positive Control Dilution Test (Started yesterday)= Back to Calendar ==Buffers== '''KLN Mix''' {| cl...") |
>Djacobse |
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| height="15" valign="bottom" | Amp Ligase Buffer | | height="15" valign="bottom" | Amp Ligase Buffer | ||
| align="center" valign="bottom" | 10X | | align="center" valign="bottom" | 10X | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 3 | ||
|- style="background-color:#D9D9D9;font-size:12pt" | |- style="background-color:#D9D9D9;font-size:12pt" | ||
| height="15" valign="bottom" | dNTPs | | height="15" valign="bottom" | dNTPs | ||
| align="center" valign="bottom" | 100 uM | | align="center" valign="bottom" | 100 uM | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 3 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Hemo Klentaq | | height="15" valign="bottom" | Hemo Klentaq | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 6 | ||
|- style="background-color:#D9D9D9;font-size:12pt" | |- style="background-color:#D9D9D9;font-size:12pt" | ||
| height="15" valign="bottom" | Amp Ligase | | height="15" valign="bottom" | Amp Ligase | ||
| align="center" valign="bottom" | 5 U/uL | | align="center" valign="bottom" | 5 U/uL | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 3 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 15 | ||
|} | |} | ||
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<ol type="A"> | <ol type="A"> | ||
<li>Prepare 20 uL KLN mix</li> | <li>Prepare 20 uL KLN mix</li> | ||
<li>Add 2 uL KLN mix to 30 uL of reaction</li> | |||
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-8-28|tomorrow]] </li> | |||
<li>Add 2 uL KLN mix to | |||
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-8- | |||
</ol></ol> | </ol></ol> | ||
[[Category:ComboLock]] [[Category:20160826]] | [[Category:ComboLock]] [[Category:20160826]] |
Latest revision as of 20:26, 29 August 2016
Positive Control Dilution Test (Started yesterday)[edit]
Buffers[edit]
KLN Mix
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 |
Protocol[edit]
- Latch and Padlock Hybridization
- Suspend 1 uL (4 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL oligo per sample to bead solution; incubate at RT for 5 min
- Apply magnet for 30 sec and remove supernatant
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- 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
- Dissolve target in 50 uL wash buffer and 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 20 uL KLN mix
- Add 2 uL KLN mix to 30 uL of reaction
- Incubate at 55C overnight; continued tomorrow