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=Circularization Optimization Testing (Started [[Daniel:Notebook/ComboLock/2016-10-31|Monday]])= | |||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
==Library Prep== | ==Library Prep (Started [[Daniel:Notebook/ComboLock/2016-11-2|Yesterday]])== | ||
<ol start="3"> | <ol start="3"> | ||
Line 13: | Line 14: | ||
<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> | </ol></ol> | ||
===Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4BD97;font-size:12pt;font-weight:bold" align="center" | |||
| width="165" height="26" | Sample | |||
| width="65" | ng/uL | |||
| width="65" | ug in 30 uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phusion-RCA Product | |||
| align="center" align="center" valign="bottom" | 58.8 | |||
| align="center" align="center" valign="bottom" | 1.8 | |||
|} | |||
=Antibody Binding Test= | |||
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="120" height="30" | Sample | |||
| width="75" | 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> | |||
<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></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 for 2 hours</li> | |||
<li>Heat kill by incubating at 94C for 5 minutes</li></ol> | |||
<li>Rolling Circle Amplification (Samples 1,3,5,7; Hold samples 2,4,6,8 at 4C)</li> | |||
<ol type="A"> | |||
<li>Prepare 8.1X Master mix according to table below</li> | |||
<li>Add sample to new reaction tubes according to following table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="160" height="30" | Reagent | |||
| width="85" | uL Added | |||
| width="85" | Master Mix (10.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Template | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" valign="bottom" | NA | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | RCA Primer (100 uM) | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 25.5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (1 mM) | |||
| align="center" align="center" valign="bottom" | 0.8 | |||
| align="center" align="center" valign="bottom" | 8.16 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 20.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 10.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 8.7 | |||
| align="center" align="center" valign="bottom" | 88.74 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |||
| align="center" align="center" valign="bottom" | 153 | |||
|} | |||
<li>Incubate at 37C for 3 hours</li> | |||
<li>Incubate at 65C for 10 minutes</li> | |||
<li>Hold at 10C; continued [[Daniel:Notebook/ComboLock/2016-11-4|tomorrow]]</li></ol> | |||
</ol> | |||
===Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |||
| width="170" height="30" | Reagent | |||
| width="65" | Stock Conc | |||
| width="65" | Final Amount | |||
| width="65" | 1x Vol (uL) | |||
| width="100" | MM Vol (10.5x) (uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | NAD+ | |||
| align="center" valign="bottom" | 5 mM | |||
| align="center" valign="bottom" | 40 nmol | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 84 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | dNTP | |||
| align="center" valign="bottom" | 1 mM | |||
| align="center" valign="bottom" | 600 pmol | |||
| align="center" align="center" valign="bottom" | 0.6 | |||
| align="center" align="center" valign="bottom" | 6.3 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Betaine | |||
| align="center" valign="bottom" | 5 M | |||
| align="center" valign="bottom" | 15 umol | |||
| align="center" align="center" valign="bottom" | 3 | |||
| align="center" align="center" valign="bottom" | 31.5 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X AmpLigase Buffer | |||
| align="center" valign="bottom" | 10X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 21 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase | |||
| align="center" valign="bottom" | 5 U/uL | |||
| align="center" valign="bottom" | 10 U | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 21 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" valign="bottom" | Phusion HF DNA Polymerase | |||
| align="center" valign="bottom" | 2000 U/mL | |||
| align="center" valign="bottom" | 6.4U | |||
| align="center" align="center" valign="bottom" | 3.2 | |||
| align="center" align="center" valign="bottom" | 33.6 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nf H2O | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | 1.2 | |||
| align="center" align="center" valign="bottom" | 12.6 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Total | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | 20 | |||
| align="center" align="center" valign="bottom" | 210 | |||
|} | |||
[[Category:ComboLock]] [[Category:20161103]] [[Category:20161031]] |
Latest revision as of 19:25, 8 November 2016
Circularization Optimization Testing (Started Monday)[edit]
Library Prep (Started Yesterday)[edit]
- 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
Results[edit]
Sample | ng/uL | ug in 30 uL |
Phusion-RCA Product | 58.8 | 1.8 |
Antibody Binding Test[edit]
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
- 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 for 2 hours
- Heat kill by incubating at 94C for 5 minutes
- Rolling Circle Amplification (Samples 1,3,5,7; Hold samples 2,4,6,8 at 4C)
- Prepare 8.1X Master mix according to table below
- Add sample to new reaction tubes according to following table
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes
- Hold at 10C; continued tomorrow
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 |
Results[edit]
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (10.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 84 |
dNTP | 1 mM | 600 pmol | 0.6 | 6.3 |
Betaine | 5 M | 15 umol | 3 | 31.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 21 |
Amp Ligase | 5 U/uL | 10 U | 2 | 21 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 33.6 |
nf H2O | 1.2 | 12.6 | ||
Total | 20 | 210 |