Daniel:Notebook/ComboLock/2016-11-15

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BSA Beads Test[edit]

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Given the results from the primers test and most recent cells test, it seems that using the primer from Chris with RCA has provided a more consistent positive control. I'm going to repeat the experiment from November 3rd regarding the BSA-strep beads.

Protocol[edit]

Sample BSA Ab BSA-Biotin Dilution Dilution Amount
Sample 1 (AB) X X 0 2 ug
Sample 2 (AB) X   NA None
Sample 3 (AB)   X 0 2 ug
Sample 4 (AB) X X 2 20 ng
Sample 5 (AB) X X 4 200 pg


  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 2 mg/mL biotin-BSA 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 2 uL of BSA Ab-AB mix (75 ug/uL stock) and 3 uL bead wash buffer into a 0.2 mL tube
    2. Incubate at RT for 1.5 hours with agitation
    3. Add 100 uL of wash buffer
    4. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    5. Repeat wash step above
    6. Add 100 uL cold Low Salt Buffer
    7. apply magnet and remove supernatant
  3. Latch and Padlock Hybridization
    1. Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0201 to each sample to a fresh 0.2 mL tube
      1. Tech Rep A-Latch0001
      2. Tech Rep B-Latch0002
    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. Add 100 uL wash buffer
    7. Apply magnet and remove supernatant; Repeat wash step above
    8. Add 100 uL cold Low Salt Buffer and vortex to suspend
    9. Apply magnet and remove supernatant
    10. Resuspend with 20 uL 1x Amp Ligase buffer
  4. Circularization
    1. Prepare Phusion mix
    2. Phusion Mix
      Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (10.2x) (uL)
      NAD+ 5 mM 40 nmol 8 81.6
      dNTP 1 mM 600 pmol 0.6 6.12
      Betaine 5 M 15 umol 3 30.6
      10X AmpLigase Buffer 10X 1X 2 20.4
      Amp Ligase 5 U/uL 10 U 2 20.4
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 32.64
      nf H2O     1.2 12.24
      Total     20 204
    3. Add 20 uL Phusion mix to 20 uL of reaction
    4. Incubate at 55C for 2 hours
    5. Heat kill by incubating at 94C for 5 minutes; tomorrow

Library Prep[edit]

  1. Size Select Gel
    1. Mix 20 uL sample and 4 uL 6x loading dye into separate tubes for each sample (Samples 1AA, 1BA, 2AB, 2BB)
    2. Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
    3. Aliquot 24 uL per sample/ladder lane into each well
    4. Run gel for 23 minutes at 225V
    5. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    6. Rinse gel and image in gel doc
    7. Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
    8. Image in gel doc
    9. Centrifuge at 12000 rpm for 1.5 minutes
    10. Add 500 uL TE to each sample
    11. Incubate 4 hours at 37C with vigorous shaking;
    12. Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
    13. Extract supernatant and add to nanosep column
    14. Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
  2. Ethanol Precipitation
    1. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
    2. Incubate at -80C overnight; continued tomorrow