Daniel:Notebook/ComboLock/2017-1-23

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Circularization Test[edit]

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Since the results from the last experiment seemed to indicate that RCA hasn't been working for me, I'm going to try RCA again using the old positive amplicon systems. For examples see Oct-5-2016 and Sept-30-2016. I'll be using PCAmp4

Sample Matrix[edit]

Tube No Molecules Available Amount
0 (AB) 1.00E+11 1 pmol
2 (AB) 1.00E+09 10 fmol
4 (AB) 1.00E+07 100 amol
6 (AB) 1.00E+05 1 amol

Protocol[edit]

  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. Prep 8.2X Padlock0501 mixture buffer
      1. 16.4 uL 10 uM Padlock0501
      2. 16.4 uL 10X Amp Ligase Buffer
      3. 131.2 uL nfH2O
    7. Heat probes to 95C 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 vigorous shaking
  2. Circularization
    1. Prepare 8.5X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (8.5x) (uL)
      NAD+ 5 mM 40 nmol 8 68
      dNTP 1 mM 600 pmol 0.6 5.1
      Betaine 5 M 15 umol 3 25.5
      10X AmpLigase Buffer 10X 1X 2 17
      Amp Ligase 5 U/uL 10 U 2 17
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 27.2
      nf H2O     1.2 8.4
      Total     20 140
    3. Add 20 uL phusion mix to 20 uL reaction
    4. Incubate at 55C for 4 hours

    Continued tomorrow