Daniel:Notebook/ComboLock/2016-11-22

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Circularization Test 2[edit]

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I'm going to try doing another circularization test but this time using a modified protocol for latch/padlock hybridization. I'm going to use the positive control C Probe system for simplicity.

Sample Matrix[edit]

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No C Probe

Protocol[edit]

  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 following to a fresh 0.2 mL tube
      1. 12.2 uL Latch0001, 10 uM stock
      2. 12.2 uL Padlock0201, 10 uM stock
      3. 12.2 uL Amp10X buffer
      4. 85.4 uL nfH2O
    2. Add 20 uL probe mix to each sample; incubate 20C for 4 min
    3. Incubate at 95C for 5 min
    4. Incubate at 60C for 1.5 hours
  4. Circularization
    1. Prepare 7X uL Phusion mix
    2. Add 20 uL Phusion mix to 20 uL of reaction
    3. Incubate at 60C for 2 hours

Continued tomorrow

Buffers[edit]

Phusion Mix

Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (7x) (uL)
NAD+ 5 mM 40 nmol 8 56
dNTP 1 mM 600 pmol 0.6 4.2
Betaine 5 M 15 umol 3 21
10X AmpLigase Buffer 10X 1X 2 14
Amp Ligase 5 U/uL 10 U 2 14
Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 22.4
nf H2O     1.2 8.4
Total     20 140