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" | | |||
| 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" | | |||
| 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 | |||
|} | |||
<ol> | <ol> |
Revision as of 22:40, 2 November 2016
Circularization Optimization Testing (Started Monday)
Library Prep (Started Yesterday)
- Ethanol Precipitation
- Centrifuge for 25 minutes at 12000 rpm at 4C
- Remove supernatant and add 750 uL 70% EtOH
- Centrifuge for 12 minutes at 12000 rpm at 4C
- Remove supernatant and spin in vacuum centrifuge for 10 minutes
- 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 |
- Template-Bead Binding
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL 2 mg/mL biotin-BSA per sample to bead solution; incubate at RT for 5 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- C Probe Hybridization
- Combine 2 uL of BSA Ab-AB mix (75 ug/uL stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Incubate at RT for 1.5 hours with agitation
- Add 100 uL of wash buffer; take 10 uL as a time point
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer; take 10 uL as a time point
- apply magnet and remove supernatant
- Latch and Padlock Hybridization
- Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0201 to each sample to a fresh 0.2 mL tube
- Tech Rep A-Latch0001
- Tech Rep B-Latch0002
- Tech Rep C-LatchX04
- Heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C with intermittent mixing
- Add 100 uL wash buffer; take 10 uL as a time point
- Apply magnet and remove supernatant; Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; take 10 uL as a time point
- Apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 55C overnight; continued tomorrow