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=Cell Test with RCA (Started [[Daniel:Notebook/ComboLock/2016-11-7|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol start="4"> <li>Exonuclease Digestion</li> <ol type="A"> <li>Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 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>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 8.1X Master mix according to table below</li> <li>Add sample to new reaction tubes according to following 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="170" height="30" | Reagent | width="65" | uL Added | width="95" | Master Mix A (2.2X) | width="95" | Master Mix B (2.2X) | width="95" | Master Mix C (8.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 11 | align="center" align="center" valign="bottom" | 11 | align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer (100 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 5.5 | align="center" align="center" valign="bottom" | 5.5 | align="center" align="center" valign="bottom" | 20.5 |- style="font-size:12pt" | height="15" valign="bottom" | RCA Primer Name | align="center" valign="bottom" | NA | align="center" valign="bottom" | RCA-CL-v01 | align="center" valign="bottom" | Chris' Primer | align="center" valign="bottom" | RCA-CL-v01 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="center" align="center" valign="bottom" | 0.8 | align="center" align="center" valign="bottom" | 1.76 | align="center" align="center" valign="bottom" | 1.76 | align="center" align="center" valign="bottom" | 6.56 |- style="font-size:12pt" | height="15" valign="bottom" | 10X Buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 4.4 | align="center" align="center" valign="bottom" | 4.4 | align="center" align="center" valign="bottom" | 16.4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 2.2 | align="center" align="center" valign="bottom" | 2.2 | align="center" align="center" valign="bottom" | 8.2 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 Company | align="center" valign="bottom" | NA | align="center" valign="bottom" | NEB | align="center" valign="bottom" | NEB | align="center" valign="bottom" | Epicentre |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 8.7 | align="center" align="center" valign="bottom" | 19.14 | align="center" align="center" valign="bottom" | 19.14 | align="center" align="center" valign="bottom" | 71.34 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 44 | align="center" align="center" valign="bottom" | 123 |} <li>Incubate at 37C for 3 hours</li> <li>Heat kill enzyme by incubating at 65C for 10 minutes</li> </ol> <li>qPCR</li> <ol type="A"> <li>Make master mix according to following recipe</li> <ol type="a"> <li>291.1 uL nfH2O</li> <li>227.5 uL SYBR Fast</li> <li>9.1 uL 10 uM AmpF</li> </ol> <li>Add 47 uL master mix to each well</li> <li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) 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="100" | Condition | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="85" | 2X Kapa SYBR qPCR MM | width="85" | 10 uM Forward Primer | width="85" | 10 uM Reverse Primer | width="65" | H2O | width="85" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1AC |style="font-weight:bold" align="center" | Experiment | align="center" | A1 | align="center" align="center" | 20 | 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 1BC |style="font-weight:bold" align="center" | Experiment | align="center" | A2 | align="center" align="center" | 20 | 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 2AC |style="font-weight:bold" align="center" | Experiment | align="center" | A3 | align="center" align="center" | 21 | 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 2BC |style="font-weight:bold" align="center" valign="bottom" | Experiment | align="center" | A4 | align="center" align="center" | 21 | 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 3AC |style="font-weight:bold" align="center" | No C Probes | align="center" | A5 | align="center" align="center" | 22 | 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 3BC |style="font-weight:bold" align="center" | No C Probes | align="center" | A6 | align="center" align="center" | 22 | 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 4AC |style="font-weight:bold" align="center" | No Cells | align="center" | A7 | align="center" align="center" | 23 | 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 4BC |style="font-weight:bold" align="center" valign="bottom" | No Cells | align="center" | A8 | align="center" align="center" | 23 | 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 1AA |style="font-weight:bold" align="center" | Exp-Epicentre | align="center" | H1 | align="center" align="center" | 24 | 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 1BA |style="font-weight:bold" align="center" | Exp-Epicentre | align="center" | H2 | align="center" align="center" | 24 | 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 2AB |style="font-weight:bold" align="center" | Exp-Primer2 | align="center" | H3 | align="center" align="center" | 25 | 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 2BB |style="font-weight:bold" align="center" valign="bottom" | Exp-Primer2 | align="center" | H4 | align="center" align="center" | 25 | 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 |style="font-weight:bold" align="center" align="center" | | align="center" | H5 | align="center" align="center" | 26 | 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 120 uL TBE, 30 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 22 minutes at 250V</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:20161108-qPCR-RCACellTest.png|qPCR curve File:2016-11-09CellRCA-Gel1.png|Gel image 1 File:2016-11-09CellRCA-Gel2.png|Gel image 2 </gallery> =Controls Test= Testing the control samples that I have. I have used several positive controls and received no results when I should. So I'm testing the various control samples to see what comes out of it. <ol> <li>qPCR</li> <ol type="A"> <li>Make master mix according to following recipes</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="100" height="30" | Master Mix | width="100" | Primer Pair | width="120" | uL Forward Primer 10 uM) | width="120" | uL Reverse Primer (10 uM) | width="150" | uL 2X Kapa SYBR Fast Master Mix | width="85" | uL nfH2O |- style="font-size:12pt" | height="15" valign="bottom" | MM1 (4.1X) | align="center" valign="bottom" | p24/p4RC | align="center" align="center" | 4.1 | align="center" align="center" | 4.1 | align="center" align="center" | 102.5 | align="center" align="center" | 86.1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | MM2 (4.1X) | align="center" valign="bottom" | p6/p12RC | align="center" align="center" | 4.1 | align="center" align="center" | 4.1 | align="center" align="center" | 102.5 | align="center" align="center" | 86.1 |- style="font-size:12pt" | height="15" valign="bottom" | MM3 (3.1X) | align="center" valign="bottom" | p24/p12RC | align="center" align="center" | 3.1 | align="center" align="center" | 3.1 | align="center" align="center" | 77.5 | align="center" align="center" | 65.1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | MM4 (3.1X) | align="center" valign="bottom" | p6/p4RC | align="center" align="center" | 3.1 | align="center" align="center" | 3.1 | align="center" align="center" | 77.5 | align="center" align="center" | 65.1 |} <li>Add 47 uL master mix to each well</li> <li>Add 2 uL sample according to table in following section</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 x35</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 120 uL TBE, 30 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 22 minutes at 250V</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:20161108-qPCR-PosControlPrimers.png|qPCR curve, probe oligos File:20161108-qPCR-AbOligoPrimers.png|qPCR curve-BSA Ab-congjugated oligos File:2016-11-09-Primers-Gel1.png|Gel Image 1 File:2016-11-09-Primers-Gel2.png|Gel Image 2 </gallery> ===qPCR Sample Table=== {| 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="90" | Primers | width="56" | Master Mix | width="65" | Lane | width="71" | Expected Product Size (bp) | width="65" | Sample Vol (uL) | width="105" | 2X Kapa SYBR qPCR MM | width="85" | 10 uM Forward Primer | width="85" | 10 uM Reverse Primer | width="85" | H2O | width="85" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | BSA-PA |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | MM1 | align="center" | A1 | align="center" align="center" | 44 | 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" | BSA-PB |style="font-weight:bold" align="center" | p6/p12RC |style="font-weight:bold" align="center" | MM2 | align="center" | A2 | align="center" align="center" | 44 | 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" | BSA-PA |style="font-weight:bold" align="center" | p24/p12RC |style="font-weight:bold" align="center" | MM3 | align="center" | A3 | align="center" align="center" | 0 | 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" | BSA-PB |style="font-weight:bold" align="center" | p6/p4RC |style="font-weight:bold" align="center" | MM4 | align="center" | A4 | align="center" align="center" | 0 | 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" | BSA Sample |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | MM1 | align="center" | A5 | align="center" align="center" | 44 | 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" | BSA Sample |style="font-weight:bold" align="center" valign="bottom" | p6/p12RC |style="font-weight:bold" align="center" valign="bottom" | MM2 | align="center" | A6 | align="center" align="center" | 44 | 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" | BSA Sample |style="font-weight:bold" align="center" | p24/p12RC |style="font-weight:bold" align="center" | MM3 | align="center" | A7 | align="center" align="center" | 0 | 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" | BSA Sample |style="font-weight:bold" align="center" valign="bottom" | p6/p4RC |style="font-weight:bold" align="center" valign="bottom" | MM4 | align="center" | A8 | align="center" align="center" | 0 | 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" | PCC-C1 |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | MM1 | align="center" | H1 | align="center" align="center" | 44 | 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" | PCC-C1 |style="font-weight:bold" align="center" | p6/p12RC |style="font-weight:bold" align="center" | MM2 | align="center" | H2 | align="center" align="center" | 0 | 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 |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | MM1 | align="center" | H3 | align="center" align="center" | 0 | 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" | NTC |style="font-weight:bold" align="center" valign="bottom" | p24/p12RC |style="font-weight:bold" align="center" valign="bottom" | MM2 | align="center" | H4 | align="center" align="center" | 0 | 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 |style="font-weight:bold" align="center" | p6/p12RC |style="font-weight:bold" align="center" | MM3 | align="center" | H5 | align="center" align="center" | 0 | 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" | NTC |style="font-weight:bold" align="center" | p6/p4RC |style="font-weight:bold" align="center" | MM4 | align="center" | H6 | align="center" align="center" | 0 | 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 |} [[Category:ComboLock]] [[Category:20161107]]
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