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=Positive Control Amplicon Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Buffer Prep== '''Wash/Binding 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 10 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" | 3.33 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" | 400 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" | 20 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" | 6.25 mL |} '''Elution 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 1 mL (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Tris-HCl | align="center" valign="bottom" | 500 mM | align="center" valign="bottom" | 10 mM | align="center" align="center" valign="bottom" | 50 | align="center" align="center" valign="bottom" | 20 |- style="background-color:#D9D9D9;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" align="center" valign="bottom" | 2 |- style="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" align="center" valign="bottom" | 978 |} '''Low Salt 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 1 mL (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NaCl | align="center" valign="bottom" | 1.5 M | align="center" valign="bottom" | 0.15 M | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 100 |- 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" align="center" valign="bottom" | 40 |- 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" align="center" valign="bottom" | 2 |- 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" align="center" valign="bottom" | 858 |} '''KLN Mix''' {| 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="180" height="31" | Reagent | width="65" | Stock | width="65" | uL added |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase Buffer | align="center" valign="bottom" | 10X | align="center" align="center" valign="bottom" | 2 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | dNTPs | align="center" valign="bottom" | 100 uM | align="center" align="center" valign="bottom" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | Hemo Klentaq | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 4 |- 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" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 10 |} ==Buffers== '''KLN Mix''' {| 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="187" height="40" | Reagent | width="65" | Stock | width="65" | uL added |- style="font-size:12pt" | height="15" 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 |} ==Protocol== <ol> <li>Latch and Padlock Hybridization</li> <ol type="A"> <li>Suspend 1 uL (4 ug) beads per sample in 100 uL wash buffer (total)</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | width="49" height="31" | Tube | width="121" | Biotin-Amplicon | width="54" | uL beads added | width="105" | uL biotin probe added |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 1 | align="center" valign="bottom" | Amp1 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 10 |- style="background-color:#D9D9D9;font-size:12pt" | align="center" height="15" valign="bottom" | 2 | align="center" valign="bottom" | Amp3 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 10 |- style="font-size:12pt" | align="center" height="30" valign="bottom" | 3 | align="center" valign="bottom" | PCAmp2 (C2) | align="center" align="center" valign="bottom" | 1 | align="center" valign="bottom" | 2 (dilute to 1 pmol/uL) |} <li>Apply magnet for 30 sec and remove supernatant</li> <li>Add 2 uL oligo per sample to bead solution (see above table); incubate at RT for 5 min</li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> <li>Mix together samples according to table in 0.2 mL tubes; use Padlock0001-68 </li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | width="49" height="31" | Sample No | width="180" | Sample Name | width="95" | LatchX2 (300 ng - 2 uL 10 uM) | width="94" | LatchX3 (300 ng - 2 uL 10 uM) | width="54" | PCAmp1 | width="51" | PCAmp3 | width="100" | Expected Product |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 1 | valign="bottom" | Amp1 Test | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | Amp1 |- style="background-color:#D9D9D9;font-size:12pt" | align="center" height="15" valign="bottom" | 2 | valign="bottom" | Amp3 Test | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | X | align="center" valign="bottom" | Amp3 |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 3 | valign="bottom" | LatchX2 Specificity | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | Amp1 |- style="background-color:#D9D9D9;font-size:12pt" | align="center" height="15" valign="bottom" | 4 | valign="bottom" | LatchX3 Specificity | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | Amp3 |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 5 | valign="bottom" | No Template | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | None |- style="background-color:#D9D9D9;font-size:12pt" | align="center" height="15" valign="bottom" | 6 | valign="bottom" | No Latch | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | None |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 7 | valign="bottom" | No Padlock | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" valign="bottom" | None |- style="background-color:#D9D9D9;font-size:12pt" | align="center" height="15" valign="bottom" | 8 | valign="bottom" | PCAmp2 Only | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | | align="center" valign="bottom" | None |} <li>Dissolve target in 50 uL wash buffer and 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</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 20 uL KLN mix</li> <li>Prepare 4.1x master mix of Phusion mix</li> <li>Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5</li> <li>Add 2 uL KLN mix to 20 uL of reaction for sample 1</li> <li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-8-19|tomorrow]] </li> </ol></ol> [[Category:ComboLock]] [[Category:20160818]]
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