Daniel:Notebook/ComboLock/2016-8-27

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Revision as of 20:11, 27 August 2016 by >Djacobse (Created page with "=Positive Control Dilution Test (Started yesterday)= Back to Calendar ==Buffers== '''KLN Mix''' {| cl...")
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Positive Control Dilution Test (Started yesterday)

Back to Calendar

Buffers

KLN Mix

Reagent Stock uL added
Amp Ligase Buffer 10X 2
dNTPs 100 uM 2
Hemo Klentaq NA 4
Amp Ligase 5 U/uL 2
nfH2O NA 10

Protocol

  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. Prepare 4.1x master mix of Phusion mix
    3. Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
    4. Add 2 uL KLN mix to 20 uL of reaction for sample 1
    5. Incubate at 55C overnight; continued tomorrow