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=Antibody Stage 2 Test (Started [[Daniel:Notebook/ComboLock/2016-6-27|yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol start="2"> <li>Latch and Padlock Hybridization</li> <ol type="A" start="5"> <li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li> </ol> <li>Exonuclease Digestion</li> <ol type="A"> <li>Prepare exonuclease I/III mix by mixing 2 uL exonuclease I (20 units/μL) and 10 uL exonuclease III (100 units/μL) in 1:1 ratio</li> <li>Add 2 μL of exonuclease I/III mix to reaction</li> <li>Mix the reaction by swirling pipette around the well 5 times</li> <li>Incubate reaction at 37 ºC for 2 hours</li> <li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> </ol> <li>qPCR</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:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | width="85" height="45" | Sample | width="65" | Lane | width="65" | Batch | width="65" | AmpR Index | width="65" | Cells | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AmpF 6.4 | width="65" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 | align="center" valign="bottom" | A1 | align="center" valign="bottom" | B1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 2 | align="center" valign="bottom" | A2 | align="center" valign="bottom" | B2 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 | align="center" valign="bottom" | A3 | align="center" valign="bottom" | B3 | align="center" align="center" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 | align="center" valign="bottom" | A4 | align="center" valign="bottom" | B4 | align="center" align="center" valign="bottom" | 4 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 5 | align="center" valign="bottom" | A5 | align="center" valign="bottom" | B5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 6 | align="center" valign="bottom" | A6 | align="center" valign="bottom" | B6 | align="center" align="center" valign="bottom" | 6 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |} <li>Master Mix</li> <ol type="a"> <li>165 uL KAPA SyberFast MM</li> <li>115.5 uL nfH2O</li> <li>3.3 uL AmpF 6.4</li></ol> <li>Add 43 uL Master Mix to each well</li> <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 x20</li> <li>72C 2 min</li> <li>16C hold</li> </ol> </ol> ===qPCR Results=== [[Image:VIM-Ab-LPTest_Results_20160628.png|500px]] ==qPCR Repeat== <ol> <li>qPCR</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:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | width="85" height="45" | Sample | width="65" | Lane | width="65" | Batch | width="65" | AmpR Index | width="65" | Cells | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AmpF 6.4 | width="65" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 | align="center" valign="bottom" | A1 | align="center" valign="bottom" | B1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 2 | align="center" valign="bottom" | A2 | align="center" valign="bottom" | B2 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 | align="center" valign="bottom" | A3 | align="center" valign="bottom" | B3 | align="center" align="center" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 | align="center" valign="bottom" | A4 | align="center" valign="bottom" | B4 | align="center" align="center" valign="bottom" | 4 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 5 | align="center" valign="bottom" | A5 | align="center" valign="bottom" | B5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 6 | align="center" valign="bottom" | A6 | align="center" valign="bottom" | B6 | align="center" align="center" valign="bottom" | 6 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 7 | align="center" valign="bottom" | A7 | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 7 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 19.5 | align="center" align="center" valign="bottom" | 50 |} <li>Master Mix</li> <ol type="a"> <li>192.5 uL KAPA SyberFast MM</li> <li>134.75 uL nfH2O</li> <li>3.85 uL AmpF 6.4</li></ol> <li>Add 43 uL Master Mix to each well</li> <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 x20</li> <li>72C 2 min</li> <li>16C hold</li> </ol> </ol> ===Results=== <gallery perrow=2 heights=200px widths=200px> File:VIM-Ab-LPTest2_Results_20160628.png|qPCR Results File:2016-06-28-CombolockAb-LP-Test2.jpg|Gel Results </gallery> The second qPCR confirmed that I am seeing a good deal of signal in many of the lanes that shouldn't have any. ==Trial 2== After talking with Andrew, I want to try yesterday's experiment again with a fixed number of cells (10000). This is to prevent any clogging that may occur during the protocol if there are too many cells. <ol> <li>Hybridization</li> <ol type="A"> <li>Measure cell concentration using 10 uL cell sample in 90 uL nfH2O</li> <ol type="a"><li>Human-3.9E5 cells/mL; Mouse-1.2E6 cells/mL</li></ol> <li>Extract cells to a new tube such that there are 100000 cells present</li> <ol type="a"><li>Human-256 uL; Mouse-85 uL</li></ol> <li>Pellet cells via centrifugation at 600g for 3 min</li> <li>Add 200 uL Wash Buffer (0.05% Tween 20 in 1X PBS)</li> <li>Pellet cells via centrifugation at 600g for 3 min</li> <li>Add PLA mix and probes to create hybridization solution</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | align="center" width="105" height="30" | |style="background-color:#CCC0DA;font-weight:bold" width="65" | uL PA Mix |style="background-color:#CCC0DA;font-weight:bold" width="65" | uL PB Mix |style="background-color:#CCC0DA;font-weight:bold" width="65" | uL PLA buffer |- style="font-size:12pt" | height="15" | PLA Mix E | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 4 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" | PLA Mix NC | align="center" align="center" | 0 | align="center" align="center" | 0 | align="center" align="center" | 6 |} <li>Resuspend cells in 10 uL PBS</li> <li>Mix 1 uL cell lysate with 3 uL Probe mix (10000 cells/reaction)</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | align="center" width="85" height="30" | |style="background-color:#CCC0DA;font-weight:bold" width="65" | uL PLA Mix E |style="background-color:#CCC0DA;font-weight:bold" width="65" | uL PLA Mix NC |style="background-color:#CCC0DA;font-weight:bold" width="65" | uL U87MG Cells |style="background-color:#CCC0DA;font-weight:bold" width="65" | uL 3T3 Cells |- style="font-size:12pt" | height="15" | Sample 1 | align="center" align="center" | 3 | align="center" align="center" | 0 | align="center" align="center" | 1 | align="center" align="center" | 0 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" | Sample 2 | align="center" align="center" | 3 | align="center" align="center" | 0 | align="center" align="center" | 0 | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 | align="center" align="center" | 0 | align="center" align="center" | 3 | align="center" align="center" | 1 | align="center" align="center" | 0 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 | align="center" align="center" | 0 | align="center" align="center" | 3 | align="center" align="center" | 0 | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 5 | align="center" align="center" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 |} <li>Incubate 1.5 hours at RT</li> <li>Add 200 uL 0.05% Tween 20 in PBS</li> <li>Wash twice with 0.05% Tween 20 in PBS</li> <ol type="a"><li>Pellet cells at 600xg for 3 min</li></ol> </ol> <li>Latch and Padlock Hybridization</li> <ol type="A"> <li>Resuspend pellet in 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)</li> <li> Add 1 uL of each insert/backbone (final conc. 100 nM each)</li> {| 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="65" height="30" | Batch | width="65" | Cell Sample | width="65" | Latch0001 (1 uL) | width="77" | Padlock0001-68 (1 uL) | width="80" | Padlock0001-88 (1 uL) |- style="font-size:12pt" | height="15" valign="bottom" | Batch 1 | align="center" | S1 | align="center" | X | align="center" | X | align="center" align="center" | |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Batch 2 | align="center" | S1 | align="center" | X | align="center" align="center" | | align="center" | X |- style="font-size:12pt" | height="15" valign="bottom" | Batch 3 | align="center" | S2 | align="center" | X | align="center" | X | align="center" align="center" | |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Batch 4 | align="center" | S2 | align="center" | X | align="center" align="center" | | align="center" | X |- style="font-size:12pt" | height="15" valign="bottom" | Batch 5 | align="center" | S3 | align="center" | X | align="center" | X | align="center" align="center" | |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Batch 6 | align="center" | S4 | align="center" | X | align="center" | X | align="center" align="center" | |- style="font-size:12pt" | height="15" valign="bottom" | Batch 7 | align="center" | S5 | align="center" | X | align="center" | X | align="center" valign="bottom" | |} <li>Incubate for 30 min at 37C following the sample matrix</li> <li>Wash twice with 200 uL wash buffer</li> <ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol> <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>Add 2 uL KLN mix to 20 uL of reaction</li> <li>Incubate at 55C overnight (16 hours) </li> <li>Continued [[Daniel:Notebook/ComboLock/2016-6-29|tomorrow]]</li> </ol> ===Buffers=== <ol type="A"> <li>Latch-Padlock Hybridization (LPH) Buffer</li> <ol type="a"> <li>100 nM insert/backbone oligos</li> <li>1X SSC</li> <li>40 U/mL RNasin</li> <li>PBS</li> </ol> <li>KLN Mix 20 uL total <ol type="a"> <li> 4 uL HemoKlentaq (20% v/v)</li> <li> 2 μL Ampligase (0.5 U/uL)</li> <li> 2 uL 1 mM dNTP mix (100 μM ea)</li> <ol type="i"><li>Add 10 uL 10mM dNTP mix to 90 uL nfH2O</li></ol> <li> 2 uL 1x Ampligase Buffer 10x</li> <li> 10 uL nfH2O</li> </ol> </ol> '''LPH 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="110" height="42" | Reagent | width="65" | Stock | width="65" | Final | width="65" | Dilution | width="74" | Amt in 100 uL (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Oligos | align="center" valign="bottom" | 10 uM | align="center" valign="bottom" | 100 nM | align="center" align="center" valign="bottom" | 100 | align="center" valign="bottom" | 1 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | SSC | align="center" valign="bottom" | 20X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | 20 uL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Rnasin | align="center" valign="bottom" | 40000 U/mL | align="center" valign="bottom" | 40 U/mL | align="center" align="center" valign="bottom" | 100 | align="center" valign="bottom" | 1 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | PBS | align="center" valign="bottom" | 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 10 uL |- 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" valign="bottom" | 68 uL |} [[Category:ComboLock]] [[Category:20160628]] [[Category:20160627]]
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