Daniel:Notebook/ComboLock/2016-11-3: Difference between revisions
Jump to navigation
Jump to search
>Djacobse No edit summary |
>Djacobse |
||
Line 13: | Line 13: | ||
<li>Remove supernatant and spin in vacuum centrifuge for 10 minutes</li> | <li>Remove supernatant and spin in vacuum centrifuge for 10 minutes</li> | ||
<li>Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A-C by reusing the water</li> | <li>Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A-C by reusing the water</li> | ||
</ol></ol> | |||
=Antibody Binding Test= | |||
Test for antibody binding and amplification. I'll use a similar protocol to | |||
<ol> | |||
<li>Template-Bead Binding</li> | |||
<ol type="A"> | |||
<li>Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer </li> | |||
<li>Apply magnet for 30 sec and remove supernatant</li> | |||
<li>Add 2 uL 2 mg/mL biotin-BSA per sample to bead solution; incubate at RT for 5 min</li> | |||
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | |||
</ol> | |||
<li>C Probe Hybridization</li> | |||
<ol type="A"> | |||
<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>Add 100 uL of wash buffer; '''take 10 uL as a time point'''</li> | |||
<li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li> | |||
<li>Repeat wash step above</li> | |||
<li>Add 100 uL cold Low Salt Buffer; '''take 10 uL as a time point'''</li> | |||
<li>apply magnet and remove supernatant</li> | |||
</ol> | |||
<li>Latch and Padlock Hybridization</li> | |||
<ol type="A"> | |||
<li>Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0201 to each sample to a fresh 0.2 mL tube</li> | |||
<ol type="a"><li>Tech Rep A-Latch0001</li><li>Tech Rep B-Latch0002</li><li>Tech Rep C-LatchX04</li></ol> | |||
<li>Heat at 65C for 5 minutes</li> | |||
<li>Chill on ice for 3 minutes</li> | |||
<li>Add sample to prepared strep beads; vortex to suspend</li> | |||
<li>Incubate for 30 min at 37C with intermittent mixing</li> | |||
<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>Add 100 uL cold Low Salt Buffer and vortex to suspend; '''take 10 uL as a time point'''</li> | |||
<li>Apply magnet and remove supernatant</li> | |||
<li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol> | |||
<li>Circularization</li> | |||
<ol type="A"> | |||
<li>Prepare Phusion mix</li> | |||
<li>Add 20 uL Phusion mix to 20 uL of reaction</li> | |||
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-10-28|tomorrow]] </li> | |||
</ol></ol> | </ol></ol> |
Revision as of 22:19, 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
- 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