Daniel:Notebook/ComboLock/2016-11-15: Difference between revisions

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>Djacobse
(Created page with "=BSA Beads Test= Back to Calendar Given the results from the primers test and [[Daniel:Notebook/ComboLo...")
 
>Djacobse
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<li>Combine 2 uL of BSA Ab-AB mix (75 ug/uL stock) and 3 uL bead wash buffer into a 0.2 mL tube</li>
<li>Combine 2 uL of BSA Ab-AB mix (75 ug/uL stock) and 3 uL bead wash buffer into a 0.2 mL tube</li>
<li>Incubate at RT for 1.5 hours with agitation</li>
<li>Incubate at RT for 1.5 hours with agitation</li>
<li>Add 100 uL of wash buffer; '''take 10 uL as a time point'''</li>
<li>Add 100 uL of wash buffer</li>
<li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li>
<li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li>
<li>Repeat wash step above</li>
<li>Repeat wash step above</li>
<li>Add 100 uL cold Low Salt Buffer; '''take 10 uL as a time point'''</li>
<li>Add 100 uL cold Low Salt Buffer</li>
<li>apply magnet and remove supernatant</li>
<li>apply magnet and remove supernatant</li>
</ol>
</ol>
Line 81: Line 81:
<li>Add 100 uL wash buffer; '''take 10 uL as a time point'''
<li>Add 100 uL wash buffer; '''take 10 uL as a time point'''
<li>Apply magnet and remove supernatant; Repeat wash step above</li>
<li>Apply magnet and remove supernatant; Repeat wash step above</li>
<li>Add 100 uL cold Low Salt Buffer and vortex to suspend; '''take 10 uL as a time point'''</li>
<li>Add 100 uL cold Low Salt Buffer and vortex to suspend</li>
<li>Apply magnet and remove supernatant</li>
<li>Apply magnet and remove supernatant</li>
<li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol>
<li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol>

Revision as of 17:35, 15 November 2016

BSA Beads Test

Back to Calendar

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

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; 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
    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