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=Sequential Hybridization Test (Started [[Daniel:Notebook/ComboLock/2016-7-5|yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol start="3"> <li>Circularization</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 10 uL exonuclease I (20 units/μL) and 2 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" | 22 | 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" | 23 | 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" | 24 | 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" | 25 | 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" | 26 | 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" | NTC | align="center" align="center" valign="bottom" | 27 | 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" | 19.5 | align="center" align="center" valign="bottom" | 50 |} <li>Master Mix</li> <ol type="a"> <li>165 uL KAPA SyberFast MM</li> <li>116 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=== <gallery perrow=2 heights=200px widths=200px> File:CProbe-VIM-SOD1-20160706-Sequential.png|qPCR Curve File:2016-07-06-VIM-SOD1-Seq.jpg|Gel Image </gallery> ==Protocol Part II== Since lanes 1-5 showed product, I'm going to sequence all of them. This is disheartening as MORE samples showed signal now that I changed to sequential hybridization. <ol start="5"> <li>Size Selection</li> <ol type="A"> <li>Combine 20 uL sample from each of the five samples</li> <ol type="a"><li>Add only 10 uL from lane 3 plus 10 uL TBE since lane 3 had greatest signal</li></ol> <li>Add 30 uL 6X loading dye</li> <li>Aliquot 30 uL each into 4 lanes</li> <li>2 uL ladder with 5 uL gel loading dye and 23 uL TBE buffer</li> <li>Run gel for 45 min at 250V</li> <li>Stain with 3 uL SYBR Gold for 3 min</li> <li>Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube</li> <gallery perrow=2 heights=200px widths=200px> File:|Before Image File:|After Image </gallery> <li>Centrifuge gel at 14000rpm for 1.5 minutes</li> <li>Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel</li> <li>Incubate at 37C for at least an hour with vigorous shaking</li> <li>Centrifuge at 14000rpm for 1.5 minutes</li> <li>Transfer supernatant to a nanosep column</li> <li>Centrifuge at 14000rpm for 1.5 minutes</li> <li>Transfer flow through to a 2 mL tube</li> </ol> <li>Ethanol Precipitation</li> <ol type="A"> <li>Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample</li> <li>Incubate overnight at -80C; continued [[Daniel:Notebook/ComboLock/2016-7-7|tomorrow]]</li></ol> </ol>
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