Daniel:Notebook/ComboLock/2016-10-13: Difference between revisions
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===Buffers=== | ===Buffers=== | ||
==Protocol== | ==Experiment Protocol== | ||
<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 template oligo 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 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube</li> | |||
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> | |||
<li>Add 5 uL probe mixture to beads</li> | |||
<li>Incubate at 40C for 1 hour with agitation</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; 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 Padlock0001-68 to each sample to a fresh 0.2 mL tube</li> | |||
<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>Apply magnet and remove supernatant</li> | |||
<li>Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant</li> | |||
<li>Repeat wash step above</li> | |||
<li>Add 100 uL cold Low Salt Buffer and vortex to suspend; 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 10 uL KLN mix</li> | |||
<li>Add 2 uL KLN mix to 20 uL of reaction</li> | |||
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-10-8|tomorrow]] </li> | |||
</ol></ol> | |||
[[Category:ComboLock]] [[Category:20161007]] | [[Category:ComboLock]] [[Category:20161007]] |
Revision as of 18:09, 13 October 2016
Padlock0301 Test (Started Friday 10-7)
Library Prep
- Size Select Gel
- Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
- Extract supernatant and add to nanosep column
- Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
- Ethanol Precipitation
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
- Incubate at -80C for a while
- 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
Padlock0301 Dilution Test
Stock Dilutions
Create the following dilutions of PCC Template by taking 10 uL stock and diluting with 90 uL nfH2O (note:zero is normal 10 uM stock). #6 is the highest concentration, made on Friday 10-7.
Tube No | Dilution | Molecules Available | Amount |
6 | 1.00E+06 | 1.00E+06 | 10 amol |
7 | 1.00E+07 | 1.00E+05 | 1 amol |
8 | 1.00E+08 | 1.00E+04 | 100 zmol |
9 | 1.00E+09 | 1000 | 10 zmol |
10 | 1.00E+10 | 100 | 1 zmol |
11 | 1.00E+11 | 10 | 100 ymol |
12 | 1.00E+12 | 1 | 10 ymol |
Buffers
Experiment Protocol
- 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 template oligo 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 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
- Latch and Padlock Hybridization
- Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube
- 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
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 10 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight; continued tomorrow