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=Circularization Optimization Testing (Started [[Daniel:Notebook/ComboLock/2016-10-31|Monday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Library Prep (Started [[Daniel:Notebook/ComboLock/2016-11-2|Yesterday]])== <ol start="3"> <li>Ethanol Precipitation</li> <ol type="A" start="2"> <li>Centrifuge for 25 minutes at 12000 rpm at 4C</li> <li>Remove supernatant and add 750 uL 70% EtOH</li> <li>Centrifuge for 12 minutes at 12000 rpm at 4C</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> </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]]
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