Daniel:Notebook/ComboLock/2016-8-27: Difference between revisions

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(Created page with "=Positive Control Dilution Test (Started yesterday)= Back to Calendar ==Buffers== '''KLN Mix''' {| cl...")
 
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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" | 2
| 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" | 2
| 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" | 4
| 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" | 2
| 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" | 10
| 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>Prepare 4.1x master mix of Phusion mix</li>
<li>Add 2 uL KLN mix to 30 uL of reaction</li>
<li>Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5</li>
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-8-28|tomorrow]] </li>
<li>Add 2 uL KLN mix to 20 uL of reaction for sample 1</li>
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-8-23|tomorrow]] </li>
</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]

Back to Calendar

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]

  1. Latch and Padlock Hybridization
    1. Suspend 1 uL (4 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Add 2 uL oligo per sample to bead solution; incubate at RT for 5 min
    4. Apply magnet for 30 sec and remove supernatant
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. 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
    7. Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
    8. Chill on ice for 3 minutes
    9. Add sample to prepared strep beads; vortex to suspend
    10. Incubate for 30 min at 37C with intermittent mixing
    11. Apply magnet and remove supernatant
    12. Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
    13. Repeat wash step above
    14. Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
    15. Resuspend with 20 uL 1x Amp Ligase buffer
  2. Circularization
    1. Prepare 20 uL KLN mix
    2. Add 2 uL KLN mix to 30 uL of reaction
    3. Incubate at 55C overnight; continued tomorrow