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=Circularization Optimization Testing (Started [[Daniel:Notebook/ComboLock/2016-10-31|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ===Sample Matrix=== {| 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="140" height="30" | Sample | width="105" | Polymerase | width="105" | Incubation Time | width="60" | RCA Y/N |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (AB) | align="center" valign="bottom" | Klentaq | align="center" valign="bottom" | ON | align="center" valign="bottom" | Y |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 2 (AB) | align="center" valign="bottom" | Klentaq | align="center" valign="bottom" | ON | align="center" valign="bottom" | N |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 (AB) | align="center" valign="bottom" | Klentaq | align="center" valign="bottom" | 2 hr | align="center" valign="bottom" | Y |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 4 (AB) | align="center" valign="bottom" | Klentaq | align="center" valign="bottom" | 2 hr | align="center" valign="bottom" | N |- style="font-size:12pt" | height="15" valign="bottom" | Sample 5 (AB) | align="center" valign="bottom" | Phusion | align="center" valign="bottom" | ON | align="center" valign="bottom" | Y |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 6 (AB) | align="center" valign="bottom" | Phusion | align="center" valign="bottom" | ON | align="center" valign="bottom" | N |- style="font-size:12pt" | height="15" valign="bottom" | Sample 7 (AB) | align="center" valign="bottom" | Phusion | align="center" valign="bottom" | 2 hr | align="center" valign="bottom" | Y |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 8 (AB) | align="center" valign="bottom" | Phusion | align="center" valign="bottom" | 2 hr | align="center" valign="bottom" | N |} ==Protocol== <ol start="4"> <li>Circularization (all samples)</li> <ol type="A"> <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 30 uL exonuclease I (20 units/μL) and 6 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 (Samples 1,3,5,7; Hold samples 2,4,6,8 at 4C)</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="160" height="30" | Reagent | width="85" | uL Added | width="85" | Master Mix (8.1X) |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" align="center" valign="bottom" | 5 | 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" | 20.25 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="center" align="center" valign="bottom" | 0.8 | align="center" align="center" valign="bottom" | 6.48 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 16.2 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 8.1 |- 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" | 70.47 |- 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" | 121.5 |} <li>Incubate at 37C for 3 hours</li> <li>Incubate at 65C for 10 minutes</li> <li>Hold at 10C</li></ol> <li>qPCR</li> <ol type="A"> <li>Set up reaction according to table below</li> <li>Make master mixes according to following recipes</li> <ol type="a"> <li>535.5 uL nfH2O</li> <li>637.5 uL SYBR Fast</li> <li>25.5 uL 10 uM AmpF</li> </ol> <li>Add 47 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 x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 240 uL TBE, 60 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> ===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="105" height="45" | Sample | width="105" | Condition | width="65" | RCA? | 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="65" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A |style="font-weight:bold" align="center" | K-ON-Y | align="center" | N | 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 1B |style="font-weight:bold" align="center" | K-ON-Y | align="center" | N | 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 2A |style="font-weight:bold" align="center" | K-ON-N | align="center" | N | 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 2B |style="font-weight:bold" align="center" valign="bottom" | K-ON-N | align="center" | N | 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 3A |style="font-weight:bold" align="center" | K-2H-Y | align="center" | N | 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 3B |style="font-weight:bold" align="center" | K-2H-Y | align="center" | N | 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 4A |style="font-weight:bold" align="center" | K-2H-N | align="center" | N | 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 4B |style="font-weight:bold" align="center" valign="bottom" | K-2H-N | align="center" | N | 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 5A |style="font-weight:bold" align="center" | P-ON-Y | align="center" | N | align="center" | F1 | 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:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 5B |style="font-weight:bold" align="center" | P-ON-Y | align="center" | N | align="center" | F2 | 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 6A |style="font-weight:bold" align="center" | P-ON-N | align="center" | N | align="center" | F3 | 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:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 6B |style="font-weight:bold" align="center" valign="bottom" | P-ON-N | align="center" | N | align="center" | F4 | 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" | Sample 7A |style="font-weight:bold" align="center" | P-2H-Y | align="center" | N | align="center" | F5 | align="center" align="center" | 26 | 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:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 7B |style="font-weight:bold" align="center" | P-2H-Y | align="center" | N | align="center" | F6 | align="center" align="center" | 26 | 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 8A |style="font-weight:bold" align="center" | P-2H-N | align="center" | N | align="center" | F7 | align="center" align="center" | 27 | 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:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 8B |style="font-weight:bold" align="center" valign="bottom" | P-2H-N | align="center" | N | align="center" | F8 | align="center" align="center" | 27 | 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 1A |style="font-weight:bold" align="center" | K-ON-Y | align="center" | Y | align="center" | H1 | align="center" align="center" | 28 | 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:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B |style="font-weight:bold" align="center" | K-ON-Y | align="center" | Y | align="center" | H2 | align="center" align="center" | 28 | 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 3A |style="font-weight:bold" align="center" | K-2H-Y | align="center" | Y | align="center" | H3 | align="center" align="center" | 29 | 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:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 3B |style="font-weight:bold" align="center" | K-2H-Y | align="center" | Y | align="center" | H4 | align="center" align="center" | 29 | 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 5A |style="font-weight:bold" align="center" | P-ON-Y | align="center" | Y | align="center" | H5 | align="center" align="center" | 30 | 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:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" | Sample 5B |style="font-weight:bold" align="center" | P-ON-Y | align="center" | Y | align="center" | H6 | align="center" align="center" | 30 | 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 7A |style="font-weight:bold" align="center" | P-2H-Y | align="center" | Y | align="center" | H7 | align="center" align="center" | 31 | 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:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 7B |style="font-weight:bold" align="center" | P-2H-Y | align="center" | Y | align="center" | H8 | align="center" align="center" | 31 | 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" | height="15" valign="bottom" | NTC |style="font-weight:bold" align="center" | NA | align="center" | N | align="center" | C1 | align="center" align="center" | 33 | 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 |} ===Results=== <gallery perrow=3 heights=300px widths=300px> File:20161101-qPCR-Circularization-KvsP.png|Klentaq (Blue) vs Phusion (Red) File:20161101-qPCR-Circularization-IncTime.png|Incubation Time; Overnight (Blue) vs 2 Hour (Red) File:20161101-qPCR-Circularization-RCACompare.png|Rolling Circle Amplification (3hr); Before (Red) and After (Blue) File:2016-11-01-CircularizationTest-Klentaq.png|Gel image Post-Klentaq File:2016-11-01-CircularizationTest-Phusion.png|Gel image Post-Phusion File:2016-11-01-CircularizationTest-PostRCA.png|Gel image Post-RCA File:2016-11-01-CircularizationTest-PostRCA-Edit.png|Gel image Post-RCA; Lines edit </gallery> [[Category:ComboLock]] [[Category:20161031]]
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