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=Padlock0301 Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] This is a test of the padlock0301, which replaces the AmpF/LinkerR region of the padlock with a custom primer set. This is because my experiments on [[Daniel:Notebook/ComboLock/2016-9-29|9-29]], [[Daniel:Notebook/ComboLock/2016-9-30|9-30]], [[Daniel:Notebook/ComboLock/2016-10-3|10-3]], and [[Daniel:Notebook/ComboLock/2016-10-4|10-4]], which test the v1 and v2 padlock systems using AmpF and AmpR individually. AmpF has proved particularly dirty so I have designed v3 of the padlocks without it. ==Protocol== ===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) {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | width="65" height="45" | Tube No | width="65" | Dilution | width="65" | Molecules Available | width="65" | Amount |- style="font-size:12pt" align="center" | align="center" height="15" | 0 | align="center" | 1 | align="center" | 1.00E+12 | 10 pmol |- style="background-color:#BFBFBF;font-size:12pt" align="center" | align="center" height="15" | 1 | align="center" | 10 | align="center" | 1.00E+11 | 1 pmol |- style="font-size:12pt" align="center" | align="center" height="15" | 2 | align="center" | 100 | align="center" | 1.00E+10 | 100 fmol |- style="background-color:#BFBFBF;font-size:12pt" align="center" | align="center" height="15" | 3 | align="center" | 1000 | align="center" | 1.00E+09 | 10 fmol |- style="font-size:12pt" align="center" | align="center" height="15" | 4 | align="center" | 10000 | align="center" | 1.00E+08 | 1 fmol |- style="background-color:#BFBFBF;font-size:12pt" align="center" | align="center" height="15" | 5 | align="center" | 1.00E+05 | align="center" | 1.00E+07 | 100 amol |- style="font-size:12pt" align="center" | align="center" height="15" | 6 | align="center" | 100000 | align="center" | 1.00E+06 | 10 amol |} ===Samples=== {| 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="130" height="45" | Sample | width="65" | C Probes-VIM (C1+C2) | width="65" | C Probes-SOD1 (C1+C2) | width="65" | Template | width="65" | Dilution | width="65" | Dilution Amount |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1 (ABC) | align="center" | X | align="center" | X | align="center" | X | align="center" align="center" valign="bottom" | 0 | align="center" | 10 pmol |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 2 | align="center" | X | 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 | align="center" align="center" | | align="center" align="center" | | align="center" | X | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | 10 pmol |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4 (ABC) | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | 2 | align="center" valign="bottom" | 100 fmol |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 5 (ABC) | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | 4 | align="center" valign="bottom" | 1 fmol |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 6 (ABC) | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | 6 | align="center" valign="bottom" | 10 amol |} ===Buffers=== {| 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="120" height="39" | Reagent | width="65" | Stock | width="65" | uL added |- style="font-size:12pt" | height="30" valign="bottom" | Amp Ligase Buffer | align="center" valign="bottom" | 10X | align="center" align="center" valign="bottom" | 3 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | dNTPs | align="center" valign="bottom" | 100 uM | align="center" align="center" valign="bottom" | 3 |- style="font-size:12pt" | height="15" valign="bottom" | Hemo Klentaq | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 6 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" valign="bottom" | 5 U/uL | align="center" align="center" valign="bottom" | 3 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 15 |} ===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 Padlock0301 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]]
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