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=Sensitivity and Specificity Experiments= [[Daniel:Notebook/ComboLock|Back to Calendar]] The purpose of this experiment will be to test the sensitivity and specificity of the ComboLock system using positive controls. The sample matrix is below, along with a column for rationales of why the sample exists in the first place. Note that {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="80" height="39" | Sample No | width="110" | Template | width="100" | T1 C Probes | width="100" | T2 C Probes | width="110" | T1 Amt | width="110" | T2 Amt | width="250" | Test |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 1 | align="center" valign="bottom" | T1 | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" | 20 pmol | align="center" align="center" | 0 | valign="bottom" | +++ Control |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 2 | align="center" valign="bottom" | T2 | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | X | align="center" align="center" | 0 | align="center" | 20 pmol | valign="bottom" | +++ Control-Does T2 work? |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 3 | align="center" valign="bottom" | AA | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" align="center" | 0 | align="center" align="center" | 0 | valign="bottom" | Negative Control |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 4 | align="center" valign="bottom" | BB | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" align="center" | 0 | align="center" align="center" | 0 | valign="bottom" | Negative Control |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 5 | align="center" valign="bottom" | T1 | align="center" align="center" valign="bottom" | | align="center" valign="bottom" | X | align="center" | 20 pmol | align="center" align="center" | 0 | valign="bottom" | Negative Control-Specificity |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 6 | align="center" valign="bottom" | T2 | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" align="center" | 0 | align="center" | 20 pmol | valign="bottom" | Negative Control-Specificity |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 7 | align="center" valign="bottom" | T1/T2 50/50 | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" | 10 pmol | align="center" | 10 pmol | valign="bottom" | Orthogonality |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 8 | align="center" valign="bottom" | T1/T2 10/90 | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" | 2 pmol | align="center" | 18 pmol | valign="bottom" | Sensitivity 10:1 |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 9 | align="center" valign="bottom" | T1/T2 1/99 | align="center" valign="bottom" | X | align="center" valign="bottom" | | align="center" | 200 fmol | align="center" | 19.8 pmol | valign="bottom" | Sensitivity 100:1 |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="30" align="center" valign="bottom" | 10 | align="center" valign="bottom" | T1/T2 1/9999 | align="center" valign="bottom" | X | align="center" valign="bottom" | | align="center" | 2 fmol | align="center" | 19.998 pmol | valign="bottom" | Sensitivity 10K:1 (1000 copies of transcript in 100K cells) |} ==Protocol== <ol> <li>Template dilutions</li> <ol type="A"> <li>Mix templates at appropriate ratios</li> <ol type="a"> <li>T1/T2 50/50: Mix 2 uL each oligo (10 uM each)</li> <li>T1/T2 10/90: Mix 4 uL each oligo (1 uM T1 and 10 uM T2)</li> <li>T1/T2 1/99: Mix 4 uL each oligo (100 '''nM''' T1 and 10 uM T2)</li> <li>T1/T2 1/9999: Mix 4 uL each oligo (1 '''nM''' T1 and 10 uM T2)</li> </ol> <li>To make dilutions, mix 5 uL sample in 45 uL nfH<sub>2</sub>O</li> <li>All samples are approximately 40 pmol added (2X bead loading)</li> <li>Pre-mix templates before bead binding</li> </ol> <li>Template-Bead Binding</li> <ol type="A"> <li>Suspend 2 uL (20 ug) beads per sample in 100 uL wash buffer </li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Resuspend oligos in mixture of 20 uL Dynabuffer, 16 uL nfH<sub>2</sub>O</li> <li>4 uL appropriate template oligo mix according to sample matrix(Total beads=20 ug=10 pmol available binding; 2X excess) 6X samples='''12 uL'''</li> <li>Incubate at RT for 15 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 0.2 mL tubes</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="180" height="30" | Reagent | width="100" | uL per Sample | width="110" | T1 Mix (6X) | width="95" | T2 Mix (2X) | width="95" | T1/T2 Mix (2X) |- style="font-size:12pt" | height="15" valign="bottom" | PCCA-03 (10 uM) | align="center" align="center" | 1 | align="center" align="center" | 6 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" | 2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | PCCB-03 (10 uM) | align="center" align="center" | 1 | align="center" align="center" | 6 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | PCCA-T2-03 (10 uM) | align="center" align="center" | 1 | align="center" align="center" | 0 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" | 2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | PCCB-T2-03 (10 uM) | align="center" align="center" | 1 | align="center" align="center" | 0 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | DynaBeads Wash Buffer | align="center" align="center" | 10 | align="center" align="center" | 60 | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 20 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" | 8 | align="center" align="center" | 48 | align="center" align="center" | 16 | align="center" align="center" | 12 |- style="font-size:12pt;font-weight:bold" | height="15" valign="bottom" | Total | align="center" align="center" | 20 | align="center" align="center" | 120 | align="center" align="center" | 40 | align="center" align="center" | 40 |} <li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> <li>Add 20 uL appropriate probe mixture to beads following sample matrix above</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>Repeat wash step above</li> </ol> </ol> ==Protocol-Part 2== <ol start="4"> <li>Lock and Latch Hybridization/Ligation</li> <ol type="A"> <li>Set up the following master mix</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="200" height="48" | Reagent | width="85" | Stock Conc | width="85" | Final Conc./Amt | width="85" | uL added | width="85" | Master Mix (10.2X) |- style="font-size:12pt" | height="15" valign="bottom" | T4 Ligase Reaction Buffer | align="center" | 5X | align="center" | 1X | align="center" align="center" | 4 | align="center" align="center" valign="bottom" | 40.8 |- 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" | 10.2 |- style="font-size:12pt" | height="15" valign="bottom" | Bolt Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" valign="bottom" | 10.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 13 | align="center" align="center" valign="bottom" | 132.6 |- 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" | 193.8 |} <li>Pre-anneal latch/bolt</li> <ol type="a"><li>Heat mix to 95C for 3 minutes</li> <li>Lower mix to 55C at rate of 0.2C/sec</li> <li>Incubate at 55C for 10 minutes</li></ol> <li>Add 19 uL annealed probe mix to each sample</li> <li>Incubate at 55C for 10 minutes</li> <li>Incubate at 25C for 10 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 15 min</li> <li>Heat kill enzyme by incubating at 65C for 10 minutes</li> <li>Wash beads with 100 uL Dynabuffer</li></ol> ****** <li>Padlock Hybridization</li> <ol type="A"> <li>Add 20 uL of following 10.2X Master Mix:</li> <ol type="a"> <li>10.2 uL 10 uM Padlock0601</li> <li>20.4 uL 10X Amp Ligase buffer</li> <li>173.4 uL nfH<sub>2</sub>O</li></ol> <li>Incubate at 70C for 5 min</li> <li>Incubate at 50C for 15 minutes</li> <li>Wash once with wash buffer</li> <li>Resuspend in 20 uL 1X Amp Ligase buffer</li> </ol> ****** <li>Circularization</li> <ol type="A"> <li>Make the following master 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="200" height="30" | Reagent | width="95" | Stock Conc | width="95" | Final Amount | width="95" | 1x Vol (uL) | width="95" | MM Vol (10.5x) (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" | 84 |- 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" | 6.3 |- 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" | 31.5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X AmpLigase Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 4 | align="center" align="center" | 42 |- 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" | 21 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" 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" | 33.6 |- style="font-size:12pt" | height="15" valign="bottom" | nf H2O | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 19.2 | align="center" align="center" | 201.6 |- 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" | 40 |style="font-weight:bold" align="center" align="center" | 420 |} <li>Add 40 uL circularization mix to each sample</li> <li>Incubate at 55C for 4 hours</li> <li>Incubate at 95C for 5 minutes</li> <li>Hold at 16C</li> </ol></ol> Continued [[Daniel:Notebook/ComboLock/2017-6-6|tomorrow]] [[Category:ComboLock]] [[Category:20170605]]
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