Daniel:Notebook/ComboLock/2016-10-5

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C Probe Positive Control Test[edit]

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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
  1. Template-Bead Binding
    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 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
    4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. C Probe Hybridization
    1. Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
    2. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    3. Add 5 uL probe mixture to beads
    4. Incubate at 40C for 1 hour with agitation
    5. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    6. Repeat wash step above
    7. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
  3. Latch and Padlock Hybridization
    1. 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
    2. Heat at 65C for 5 minutes
    3. Chill on ice for 3 minutes
    4. Add sample to prepared strep beads; vortex to suspend
    5. Incubate for 30 min at 37C with intermittent mixing
    6. Apply magnet and remove supernatant
    7. Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
    8. Repeat wash step above
    9. Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
    10. Resuspend with 20 uL 1x Amp Ligase buffer
  4. Circularization
    1. Prepare 10 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight; continued tomorrow