Daniel:Notebook/ComboLock/2016-11-3

From ZhangLabWiki
Jump to navigation Jump to search

Circularization Optimization Testing (Started Monday)[edit]

Back to Calendar

Library Prep (Started Yesterday)[edit]

  1. Ethanol Precipitation
    1. Centrifuge for 25 minutes at 12000 rpm at 4C
    2. Remove supernatant and add 750 uL 70% EtOH
    3. Centrifuge for 12 minutes at 12000 rpm at 4C
    4. Remove supernatant and spin in vacuum centrifuge for 10 minutes
    5. Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A-C by reusing the water

Results[edit]

Sample ng/uL ug in 30 uL
Phusion-RCA Product 58.8 1.8

Antibody Binding Test[edit]

Test for antibody binding and amplification. I'll use a similar protocol to 10-21 which uses takes time points but I'll also be using the standard beads protocol (some modifications).

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; take 10 uL as a time point
    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; take 10 uL as a time point
    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; take 10 uL as a time point
    7. Apply magnet and remove supernatant; Repeat wash step above
    8. Add 100 uL cold Low Salt Buffer and vortex to suspend; take 10 uL as a time point
    9. Apply magnet and remove supernatant
    10. Resuspend with 20 uL 1x Amp Ligase buffer
  4. Circularization
    1. Prepare Phusion mix
    2. Add 20 uL Phusion mix to 20 uL of reaction
    3. Incubate at 55C for 2 hours
    4. Heat kill by incubating at 94C for 5 minutes
  5. Rolling Circle Amplification (Samples 1,3,5,7; Hold samples 2,4,6,8 at 4C)
    1. Prepare 8.1X Master mix according to table below
    2. Add sample to new reaction tubes according to following table
    3. Reagent uL Added Master Mix (10.2X)
      Template 5 NA
      RCA Primer (100 uM) 2.5 25.5
      dNTP (1 mM) 0.8 8.16
      10X Buffer 2 20.4
      Phi29 1 10.2
      nfH2O 8.7 88.74
      Total 20 153
    4. Incubate at 37C for 3 hours
    5. Incubate at 65C for 10 minutes
    6. Hold at 10C; continued tomorrow

Results[edit]

Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (10.5x) (uL)
NAD+ 5 mM 40 nmol 8 84
dNTP 1 mM 600 pmol 0.6 6.3
Betaine 5 M 15 umol 3 31.5
10X AmpLigase Buffer 10X 1X 2 21
Amp Ligase 5 U/uL 10 U 2 21
Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 33.6
nf H2O     1.2 12.6
Total     20 210