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=Circularization Test (Started [[Daniel:Notebook/ComboLock/2017-1-23|Monday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-New Amplification Primers== These are new primers I designed. They are slightly adjusted from the locations of AmpF/AmpR. Hopefully there is less noise. <ol> <li>qPCR</li> <ol type="A"> <li>Make qPCR master mix according to following recipe</li> <ol type="a"> <li>361.2 uL nfH2O</li> <li>17.2 uL AmpF-CLv3</li> <li>17.2 uL AmpR-CLv3</li> <li>430 uL Kapa SYBR Fast</li></ol> <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample 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="100" height="45" | Sample | width="85" | Product Amt | width="85" | Pre/Post RCA | width="65" | Lane | width="65" | Sample Vol (uL) | width="85" | 2X Kapa SYBR qPCR MM | width="65" | 10 uM Forward Primer | width="65" | 10 uM Reverse Primer | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 0A | align="center" | 1pmol | align="center" | Pre | align="center" | A1 | 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 0B | align="center" | 1pmol | align="center" | Pre | align="center" | A2 | 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 | align="center" | 10 fmol | align="center" | Pre | align="center" | A3 | 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 | align="center" valign="bottom" | 10 fmol | align="center" | Pre | align="center" | A4 | 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 | align="center" | 100 amol | align="center" | Pre | align="center" | A5 | 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 4B | align="center" | 100 amol | align="center" | Pre | align="center" | A6 | 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 | align="center" | 1 amol | align="center" | Pre | align="center" | A7 | 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 6B | align="center" | 1 amol | align="center" | Pre | align="center" | A8 | 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 0AX | align="center" | 1pmol | align="center" | Post | align="center" | H1 | 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 0BX | align="center" | 1pmol | align="center" | Post | align="center" | H2 | 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 2AX | align="center" | 10 fmol | align="center" | Post | align="center" | H3 | 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 2BX | align="center" valign="bottom" | 10 fmol | align="center" | Post | align="center" | H4 | 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 4AX | align="center" | 100 amol | align="center" | Post | align="center" | H5 | 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 4BX | align="center" | 100 amol | align="center" | Post | align="center" | H6 | 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 6AX | align="center" | 1 amol | align="center" | Post | align="center" | H7 | 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 6BX | align="center" | 1 amol | align="center" | Post | align="center" | H8 | 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 |} <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 80 uL TBE, 20 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=3 heights=300px widths=300px> File:20170125-qPCR-NewAmp.png|qPCR Curve File:2017-01-25-RCATest-NewAmp-Pre.png|Gel image-Pre RCA File:2017-01-25-RCATest-NewAmp-Post.png|Gel image-Post RCA </gallery> ==RCA with Epicentre Phi29== Still have some left, so I'm going to test its effectiveness by trying to amplify the 1 amol sample. I'll use the original Epicentre concentration and a 10X dilution, since the Epicentre Phi29 is 10X more concentrated than NEB. '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#8064A2;font-size:12pt;font-weight:bold" align="center" | width="100" height="32" | Sample | width="95" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample 6AC | align="center" valign="bottom" | Epicentre-1:10 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 6BC | align="center" valign="bottom" | Epicentre-1:10 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 6AD | align="center" valign="bottom" | Epicentre |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 6BD | align="center" valign="bottom" | Epicentre |} <ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 7X Master mix according to table below</li> <li>Add 15 uL master mix to each tube</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="250" height="30" | Reagent | width="145" | uL Added | width="105" | Master Mix (4.5X) |- 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="30" valign="bottom" | RCA Primer (LLRC 10 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 11.25 |- 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" | 3.6 |- 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" | 9 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 |- 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" | 39.15 |- 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" | 63 |} <li>Incubate at 37C for 3 hours</li> <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> Held overnight at 4C; Continued [[Daniel:Notebook/ComboLock/2017-1-26|tomorrow]] ==Library Prep== Based on [[Daniel:Notebook/ComboLock/2017-1-24|yesterday]]'s results, I'm going to sequence the most successful lanes (PreRCA-1pmol; PostRCA-1 pmol, 10 fmol, and 100 amol). With the UMI now on the padlock, this should give a relative percentage of padlock capture. Each sample has a unique AmpR index. '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#8064A2;font-size:12pt;font-weight:bold" align="center" | width="120" height="32" | Sample | width="90" | Sample Amt | width="70" | Pre/Post RCA | width="90" | AmpR Index |- style="font-size:12pt" | height="15" valign="bottom" | Sample 0 (AB) | align="center" valign="bottom" | 1 pmol | align="center" valign="bottom" | Pre | align="center" align="center" valign="bottom" | 21 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 0X (AB) | align="center" valign="bottom" | 1 pmol | align="center" valign="bottom" | Post | align="center" align="center" valign="bottom" | 25 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 2X (AB) | align="center" valign="bottom" | 10 fmol | align="center" valign="bottom" | Post | align="center" align="center" valign="bottom" | 26 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 4X (AB) | align="center" valign="bottom" | 100 amol | align="center" valign="bottom" | Post | align="center" align="center" valign="bottom" | 27 |} <ol> <li>Size Select Gel</li> <ol type="A"> <li>Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)</li> <li>Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube</li> <li>Aliquot 48/24 uL per sample/ladder lane into each well</li> <li>Run gel for 23 minutes at 230V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> <li>Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube</li> <li>Image in gel doc</li> <gallery perrow=2 heights=300px widths=300px> File:2017-01-25-RCATest-SizeSelect.png|Before Image File:2017-01-25-RCATest-SizeSelect-After.png|After Image </gallery> <li>Centrifuge at 12000 rpm for 1.5 minutes</li> <li>Add 500 uL TE to each sample</li> <li>Incubate 4 hours at 37C with vigorous shaking;</li> <li>Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom</li> <li>Extract supernatant and add to nanosep column</li> <li>Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant</li></ol> <li>Ethanol Precipitation</li> <ol type="A"> <li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample</li> <li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2017-1-26|tomorrow]]</li> </ol></ol> [[Category:ComboLock]] [[Category:20170123]]
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