Daniel:Notebook/ComboLock/2016-11-3: Difference between revisions

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>Djacobse
>Djacobse
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=Antibody Binding Test=
=Antibody Binding Test=


Test for antibody binding and amplification. I'll use a similar protocol to  
Test for antibody binding and amplification. I'll use a similar protocol to [[Daniel:Notebook/ComboLock/2016-10-21|10-21]] which uses takes time points but I'll also be using the standard beads protocol (some modifications).
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center"
| width="89" height="30" | Sample
| width="56" | BSA Ab
| width="65" | BSA-Biotin
| width="65" | Dilution
| width="65" | Dilution Amount
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 1 (AB)
| align="center" | X
| align="center" | X
| align="center" align="center" valign="bottom" | 0
| align="center" | 2 ug
 
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 2 (AB)
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | None
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 3 (AB)
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" align="center" valign="bottom" | 0
| align="center" valign="bottom" | 2 ug
 
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 4 (AB)
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | 2
| align="center" valign="bottom" | 20 ng
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 5 (AB)
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | 4
| align="center" valign="bottom" | 200 pg
 
|}
 


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Revision as of 22:40, 2 November 2016

Circularization Optimization Testing (Started Monday)

Back to Calendar

Library Prep (Started Yesterday)

  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

Antibody Binding Test

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
      3. Tech Rep C-LatchX04
    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 overnight; continued tomorrow