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==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 *this is actually PCR since the eppendorf thermocycler was down and the other was in use</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="81" 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" | A5 | 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" | A1 | 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" | B7 | align="center" align="center" valign="bottom" | 7 | 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 8 | align="center" valign="bottom" | A8 | align="center" valign="bottom" | B8 | align="center" align="center" valign="bottom" | 8 | 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>200 uL KAPA SyberFast MM</li> <li>140 uL nfH2O</li> <li>4 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> <li>PAGE gel</li> <ol type="A"> <li>Set up lanes according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#E6B8B7;font-size:12pt;font-weight:bold" align="center" | width="65" height="35" | Sample | width="65" | Lane | width="65" | uL TBE Buffer | width="65" | uL loading dye | width="65" | uL sample |- style="font-size:12pt" valign="bottom" | height="15" | Ladder | align="center" | 2 | align="center" | 4 | align="center" | 2 | align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | Batch1 | align="center" | 3 | align="center" | 4 | align="center" | 2 | align="center" | 2 |- style="font-size:12pt" valign="bottom" | height="15" | Batch2 | align="center" | 4 | align="center" | 4 | align="center" | 2 | align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | Batch3 | align="center" | 5 | align="center" | 4 | align="center" | 2 | align="center" | 2 |- style="font-size:12pt" valign="bottom" | height="15" | Batch4 | align="center" | 6 | align="center" | 4 | align="center" | 2 | align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | Batch5 | align="center" | 7 | align="center" | 4 | align="center" | 2 | align="center" | 2 |- style="font-size:12pt" valign="bottom" | height="15" | Batch6 | align="center" | 8 | align="center" | 4 | align="center" | 2 | align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | Batch7 | align="center" | 9 | align="center" | 4 | align="center" | 2 | align="center" | 2 |- style="font-size:12pt" valign="bottom" | height="15" | Batch8 | align="center" | 10 | align="center" | 4 | align="center" | 2 | align="center" | 2 |} <li>Run gel for 25 minutes at 220V</li> <li>Stained with 3 uL SYBR Gold for 3 min</li></ol> <li>Size Selection</li> <ol type="A"> <li>Take 20 uL sample from B2,B3,B5, and B6 into separate tubes</li> <li>Add 4 uL 6X loading dye to each tube</li> <li>Aliquot 24 uL each into 4 lanes (last one will have 20 uL)</li> <li>5 uL ladder with 1 uL gel loading dye</li> <li>Run gel for 25 min at 220V</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:2016-06-24-CombolockTest-Stage2-SizeSelect-Before.jpg|Before Image File:2016-06-24-CombolockTest-Stage2-SizeSelect-After.jpg|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/2016-6-25|tomorrow]]</li></ol> </ol> ===Results=== '''PCR Gel Image''' [[Image:2016-06-24-CombolockTest-Stage2-FIXED.png|500px]] I had to fix the lane annotations from the top annotations, which were the originals. I forgot (see above) that I switched lanes 1 and 5, so that definitely isn't corrected on the gel. Additionally, I believe that lanes 5-8 were put in the gel backwards (long story involving PCR machines). So, fixing those you get the black box labeling, which I believe to be correct. If that is correct, the 48 bp backbone padlock doesn't work, and it looks like the 68 works better than the 88 (qualitatively). So in the future I'd recommend around a 20 bp spacer, as is used in the 68 bp backbone version. Since I have results, I want to go ahead with size selection and sequencing. [[Category:ComboLock]] [[Category:20160623]]
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