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=RCA Time Test (Started [[Daniel:Notebook/ComboLock/2017-1-27|Friday Jan 27]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Phosphorothiorate RCA Primer (Started [[Daniel:Notebook/ComboLock/2017-2-1|Yesterday]])== <ol start="2"> <li>qPCR</li> <ol type="A"> <li>Make qPCR master mix according to following recipe</li> <ol type="a"> <li>361.2 uL nfH2O</li> <li>17.2 uL AmpF6.4</li> <li>430 uL Kapa SYBR Fast</li></ol> <li>Add 47 uL master mix to each well</li> <li>Add 2 uL sample and 1 uL AmpR indexed according to 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="120" height="45" | Sample | width="80" | Sample | width="90" | Condition | width="65" | AmpR Index | width="65" | Lane | width="65" | Sample Vol (uL) | width="85" | 2X Kapa SYBR qPCR MM | width="65" | 10 uM Forward Primer | width="65" | 10 uM Reverse Primer | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1AN | align="center" | Sample | align="center" | 3 hours | align="center" align="center" | 20 | align="center" | A8 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1BN | align="center" | Sample | align="center" | 3 hours | align="center" align="center" | 20 | align="center" | A7 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3AN | align="center" | No C Probe | align="center" | 3 hours | align="center" align="center" | 21 | align="center" | A6 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 3BN | align="center" valign="bottom" | No C Probe | align="center" | 3 hours | align="center" align="center" | 21 | align="center" | A5 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1AO | align="center" | Sample | align="center" | 3 hours-Epi | align="center" align="center" | 22 | align="center" | A4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1BO | align="center" | Sample | align="center" | 3 hours-Epi | align="center" align="center" | 22 | align="center" | A3 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3AO | align="center" | No C Probe | align="center" | 3 hours-Epi | align="center" align="center" | 23 | align="center" | A2 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 3BO | align="center" valign="bottom" | No C Probe | align="center" | 3 hours-Epi | align="center" align="center" | 23 | align="center" | A1 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | NTC | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 24 | align="center" | H1 | align="center" align="center" | 0 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 23 | align="center" align="center" | 50 |} <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> ****** <li>TBE Gel</li> <ol type="A"> <li>Mix 94 uL TBE, 24 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 23 minutes at 230V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20170202-qPCR-RCATimeTest-Phosphorothiorate.png|qPCR curve File:2017-02-02-RCATimeTest-Phosphorothiorate.png|Gel image </gallery> =Version 2 Oligos Round 2= [[Daniel:Notebook/ComboLock|Back to Calendar]] Since I've been having so [[Daniel:Notebook/ComboLock/2017-1-13|much]] [[Daniel:Notebook/ComboLock/2017-1-27|trouble]] with the version 3 oligos, I want to test the v2 oligos again. ==Sample Matrix== {| 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="150" height="34" | Sample | width="120" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (AB) | align="center" valign="bottom" | Normal |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 2 (AB) | align="center" valign="bottom" | No Template |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 (AB) | align="center" valign="bottom" | No C probes |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 4 (AB) | align="center" valign="bottom" | No phosphate activation |} ==Protocol-Phase 1== <ol> <li>Phosphorylation-MirrorC Probe B (PCCB-02)</li> <ol type="A"> <li>Set up reaction according to table</li> {| 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="150" height="30" | Reagent | width="92" | Stock Conc | width="91" | Final Conc./Amount | width="65" | uL added |- style="font-size:12pt" | height="30" valign="bottom" | T4 Polynucleotide Kinase Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | ATP | align="center" | 10 mM | align="center" | 1 mM | align="center" align="center" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | PCCB-02 | align="center" | 10 uM | align="center" | 100 pmol total | align="center" align="center" | 10 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | T4 DNA Kinase | align="center" | 10 U/uL | align="center" | 10 U | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 5 |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 20 |} <li>Incubate at 37C for 30 min</li> <li>Heat kill enzyme with 15 minutes at 65C</li> </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 10 uM 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 following into a 0.2 mL tube</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt" align="center" | align="center" width="130" height="30" | Reagent |style="font-weight:bold" width="67" | uL per Sample |style="font-weight:bold" width="65" | uL Total |- style="font-size:12pt" | height="15" valign="bottom" | PCCA | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 6 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | Mirror C Probe B reaction | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 12 |- style="font-size:12pt" | height="15" valign="bottom" | Wash buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 12 |- style="background-color:#BFBFBF;font-size:12pt;font-weight:bold" | height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 30 |} <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; '''start part 4 after incubation begins'''</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> ==Protocol-Phase 2== <li>Phosphorylation-Lock oligo</li> <ol type="A"> <li>Set up reaction according to table</li> {| 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="140" height="30" | Reagent | width="92" | Stock Conc | width="91" | Final Conc./Amount | width="65" | uL added |- style="font-size:12pt" | height="30" valign="bottom" | T4 DNA Ligase Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | ATP | align="center" | 10 mM | align="center" | 1 mM | align="center" align="center" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | Lock0201 | align="center" | 10 uM | align="center" | 10 pmol total | align="center" align="center" | 10 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | T4 DNA Kinase | align="center" | 10 U/uL | align="center" | 10 U | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 5 |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 20 |} <li>Incubate at 37C for 30 min</li> <li>Heat kill enzyme with 15 minutes at 65C</li> </ol> ****** <li>Lock and Latch Hybridization/Ligation</li> <ol type="A"> <li>Set up the following master mix; '''Note: for sample 4 add un-phosphorylated Lock0201'''</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | width="150" height="30" | Reagent | width="65" | Stock Conc | width="65" | Final Conc./Amt | width="65" | uL added | width="65" | Master Mix (6.2X) | width="65" | Master Mix (2.2X) |- style="font-size:12pt" | height="45" valign="bottom" | T4 Ligase Reaction Buffer | align="center" | 5X | align="center" | 1X | align="center" align="center" | 4 | align="center" align="center" valign="bottom" | 24.8 | align="center" align="center" valign="bottom" | 8.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Latch Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" valign="bottom" | 6.2 | align="center" align="center" valign="bottom" | 2.2 |- style="font-size:12pt" | height="45" valign="bottom" | Lock Oligo Phosphate Reaction | align="center" | (5 uM Lock) | align="center" | 10 pmol | align="center" align="center" | 2 | align="center" align="center" valign="bottom" | 12.4 | align="center" valign="bottom" | *2.2* |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 12 | align="center" align="center" valign="bottom" | 86.8 | align="center" align="center" valign="bottom" | 39.2 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 19 |style="font-weight:bold" align="center" align="center" valign="bottom" | 74.4 |style="font-weight:bold" align="center" align="center" valign="bottom" | 26.4 |} '''This table had errors'''. Correct table below but I used the table above {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | width="180" height="30" | Reagent | width="65" | Stock Conc | width="65" | Final Conc./Amt | width="65" | uL added | width="85" | Master Mix (6.2X) | width="85" | Master Mix (2.2X) |- style="font-size:12pt" | height="30" valign="bottom" | T4 Ligase Reaction Buffer | align="center" | 5X | align="center" | 1X | align="center" align="center" | 4 | align="center" align="center" | 24.8 | align="center" align="center" | 8.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Latch Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" | 6.2 | align="center" align="center" | 2.2 |- style="font-size:12pt" | height="30" valign="bottom" | Lock Oligo Phosphate Reaction | align="center" | (5 uM Lock) | align="center" | 10 pmol | align="center" align="center" | 2 | align="center" align="center" | 12.4 | align="center" | *2.2* |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 12 | align="center" align="center" valign="bottom" | 74.4 | align="center" align="center" valign="bottom" | 28.6 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 19 |style="font-weight:bold" align="center" align="center" valign="bottom" | 117.8 |style="font-weight:bold" align="center" align="center" valign="bottom" | 39.6 |} <li>Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201</li> <li>Incubate at 95C for 5 min</li> <li>Lower temperature to 40C; incubate 20 minutes</li> <li>Lower the temperature to 20C; wait 30 seconds then take off incubator</li> <li>Add 1 uL T4 Ligase to each reaction</li> <li>Incubate at RT for 20 min</li> <li>Heat kill enzyme by incubating at 65C for 10 minutes</li> <li>Wash twice with 100 uL wash buffer</li></ol></ol> ==Protocol-Part 3-Padlock Hybridization and Circularization== <ol start="6"> <li>Padlock Hybridization</li> <ol type="A"> <li>Make the following master mix</li> <ol type="a"> <li>16.4 uL 10 uM Padlock0401A</li> <li>16.4 uL Amp Ligase 10X reaction buffer</li> <li>131.2 uL nfH2O</li></ol> <li>Resuspend sample in 20 uL padlock buffer</li> <li>Incubate at 37C for 10 minutes</li></ol> ****** <li>Circularization</li> <ol type="A"> <li>Prepare 8.5X uL Phusion mix</li> {| 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="140" height="30" | Reagent | width="65" | Stock Conc | width="65" | Final Amount | width="65" | 1x Vol (uL) | width="65" | MM Vol (8.5x) (uL) |- style="font-size:12pt" | height="15" valign="bottom" | NAD+ | align="center" | 5 mM | align="center" | 40 nmol | align="center" align="center" | 8 | align="center" align="center" | 68 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | dNTP | align="center" | 1 mM | align="center" | 600 pmol | align="center" align="center" | 0.6 | align="center" align="center" | 5.1 |- style="font-size:12pt" | height="15" valign="bottom" | Betaine | align="center" | 5 M | align="center" | 15 umol | align="center" align="center" | 3 | align="center" align="center" | 25.5 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | 10X AmpLigase Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 2 | align="center" align="center" | 17 |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" | 5 U/uL | align="center" | 10 U | align="center" align="center" | 2 | align="center" align="center" | 17 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | Phusion HF DNA Polymerase | align="center" | 2000 U/mL | align="center" | 6.4U | align="center" align="center" | 3.2 | align="center" align="center" | 27.2 |- style="font-size:12pt" | height="15" valign="bottom" | nf H2O | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 1.2 | align="center" align="center" | 8.4 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 20 |style="font-weight:bold" align="center" align="center" | 140 |} <li>Add 20 uL phusion mix to 20 uL reaction</li> <li>Incubate at 40C for 4 hours</li> <li>Heat kill enzyme by incubating 10 minutes at 95C</li> <li>Hold overnight at 12C</li> </ol> Continued [[Daniel:Notebook/ComboLock/2017-2-3|tomorrow]] ===Buffers=== '''Wash Buffer''' {| 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="65" height="30" | Reagent | width="65" | Stock | width="65" | Final | width="65" | Dilution | width="65" | Amt in 20 mL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NaCl | align="center" valign="bottom" | 1.5 M | align="center" valign="bottom" | 0.5 M | align="center" align="center" valign="bottom" | 3 | align="center" valign="bottom" | 6.66 mL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Tris-HCl | align="center" valign="bottom" | 500 mM | align="center" valign="bottom" | 20 mM | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 800 uL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | EDTA | align="center" valign="bottom" | 0.5M | align="center" valign="bottom" | 1 mM | align="center" align="center" valign="bottom" | 500 | align="center" valign="bottom" | 40 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | nf H2O | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | 12.5 mL |} [[Category:ComboLock]] [[Category:20170127]] [[Category:20170202]]
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