Daniel:Notebook/ComboLock/2016-9-22: Difference between revisions

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<li>Prepare 30 uL KLN mix</li>
<li>Prepare 30 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/2016-8-30|tomorrow]] </li>
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-9-23|tomorrow]] </li>
</ol></ol>
</ol></ol>
[[Category:ComboLock]] [[Category:20160922]]

Latest revision as of 00:42, 28 September 2016

PCR Testing[edit]

Back to Calendar

I'm going to do some testing with the PCR conditions to see if I can remove the off-target products. Right now I'm testing against several potential sources:

  • Extension time may have something to do with the higher MW products (500+bp)
  • Primer annealing temp may have something to do with the primer-dimer products (add analysis link)
  • Primer concentration may affect the generation of all the products, but especially the primer dimers

For reference see the size select gel from 9-5-16 which shows the three off target products that occur.

Protocol[edit]

Tube No (A-C) Dilution Molecules Available
0 1 1E11
1 10 1E10
  1. Latch and Padlock Hybridization
    1. Suspend 2 uL (8 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 30 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight; continued tomorrow