Editing
Daniel:Notebook/ComboLock/2017-1-24
(section)
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=Circularization Test (Started [[Daniel:Notebook/ComboLock/2017-1-23|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-Part 2== <ol start="3"> <li>Exonuclease Digestion</li> <ol type="A"> <li>Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)</li> <li>Add 2 uL to each sample, mix by swirling pipette tip</li> <li>Incubate for 1.5 hours at 37C</li> <li>Heat kill by incubating for 5 min at 95C</li> </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="146" height="30" | Reagent | width="65" | uL Added | width="65" | Master Mix (8.5X) |- 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="15" valign="bottom" | RCA Primer (10 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 21.25 |- 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" | 6.8 |- 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" | 17 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 8.5 |- 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" | 73.95 |- 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" | 127.5 |} <li>Incubate at 37C for 3 hours</li> <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> ==Protocol-Part 3== <li>qPCR</li> <ol type="A"> <li>Make qPCR 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="95" height="30" | Master Mix | width="120" | Primer Pair | width="85" | uL Forward Primer | width="85" | uL Reverse Primer | width="85" | uL nfH2O | width="85" | uL 2X SYBR Fast MM | width="65" | uL Total |- style="font-size:12pt" align="center" | height="15" | MM1 | AmpF/AmpR | align="center" | 17.2 | None | align="center" | 361.2 | align="center" | 430 | align="center" | 808.4 |- style="background-color:#BFBFBF;font-size:12pt" align="center" | height="30" | MM2 | primer6RC/AmpR.Ind29 | align="center" | 9.2 | align="center" | 9.2 | align="center" | 193.2 | align="center" | 230 | align="center" | 441.6 |} <li>Add 47 (MM1) or 48 (MM2) uL master mix to each well</li> <li>Add 2 uL sample and/or 1 uL appropriate Reverse Primer (1 uM) 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 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> ===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="100" | Product Amt | width="60" | Pre/Post RCA | width="120" | Primer Pair | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="65" | 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) | width="65" | Master Mix |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 0A | align="center" | 1pmol | align="center" | Pre | align="center" | AmpF/AmpR | align="center" | A1 | 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 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 0B | align="center" | 1pmol | align="center" | Pre | align="center" | AmpF/AmpR | align="center" | A2 | 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 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 2A | align="center" | 10 fmol | align="center" | Pre | align="center" | AmpF/AmpR | align="center" | A3 | 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 | align="center" | MM1 |- 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" | AmpF/AmpR | align="center" | A4 | 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 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 4A | align="center" | 100 amol | align="center" | Pre | align="center" | AmpF/AmpR | align="center" | A5 | 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 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4B | align="center" | 100 amol | align="center" | Pre | align="center" | AmpF/AmpR | align="center" | A6 | 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 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 6A | align="center" | 1 amol | align="center" | Pre | align="center" | AmpF/AmpR | align="center" | A7 | 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 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 6B | align="center" | 1 amol | align="center" | Pre | align="center" | AmpF/AmpR | align="center" | A8 | 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 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 0AX | align="center" | 1pmol | align="center" | Post | align="center" | AmpF/AmpR | align="center" | H1 | 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 | align="center" | MM1 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 0BX | align="center" | 1pmol | align="center" | Post | align="center" | AmpF/AmpR | align="center" | H2 | 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 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 2AX | align="center" | 10 fmol | align="center" | Post | align="center" | AmpF/AmpR | align="center" | H3 | 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 | align="center" | MM1 |- 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" | AmpF/AmpR | align="center" | H4 | 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 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 4AX | align="center" | 100 amol | align="center" | Post | align="center" | AmpF/AmpR | align="center" | H5 | 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 | align="center" | MM1 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4BX | align="center" | 100 amol | align="center" | Post | align="center" | AmpF/AmpR | align="center" | H6 | 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 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 6AX | align="center" | 1 amol | align="center" | Post | align="center" | AmpF/AmpR | align="center" | H7 | 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 | align="center" | MM1 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 6BX | align="center" | 1 amol | align="center" | Post | align="center" | AmpF/AmpR | align="center" | H8 | 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 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 0A | align="center" | 1pmol | align="center" | Pre | align="center" | primer6RC/AmpR | align="center" | C1 | 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 | align="center" | MM2 |- style="background-color:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" | Sample 0B | align="center" | 1pmol | align="center" | Pre | align="center" | primer6RC/AmpR | align="center" | C2 | 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 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 6A | align="center" | 1 amol | align="center" | Pre | align="center" | primer6RC/AmpR | align="center" | C3 | 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 | align="center" | MM2 |- style="background-color:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" | Sample 6B | align="center" | 1 amol | align="center" | Pre | align="center" | primer6RC/AmpR | align="center" | C4 | 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 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 0AX | align="center" | 1pmol | align="center" | Post | align="center" | primer6RC/AmpR | align="center" | C5 | 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 | align="center" | MM2 |- style="background-color:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" | Sample 0BX | align="center" | 1pmol | align="center" | Post | align="center" | primer6RC/AmpR | align="center" | C6 | 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 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 6AX | align="center" | 1 amol | align="center" | Post | align="center" | primer6RC/AmpR | align="center" | C7 | 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 | align="center" | MM2 |- style="background-color:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" | Sample 6BX | align="center" | 1 amol | align="center" | Post | align="center" | primer6RC/AmpR | align="center" | C8 | 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 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" | NA | align="center" | AmpF/AmpR | align="center" | F1 | 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" | 23 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#B1A0C7;font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" | NA | align="center" | primer6RC/AmpR | align="center" | F2 | 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" | 23 | align="center" align="center" | 50 | align="center" | MM2 |} ===Results-Curves and Gel Images=== <gallery perrow=3 heights=300px widths=300px> File:20170124-qPCR-PrePostRCA.png|qPCR Curve File:2017-01-24-RCATest-PreRCA.png|Gel image-Pre RCA File:2017-01-24-RCATest-PostRCA.png|Gel image-Post RCA File:|qPCR Curve File:2017-01-24-RCATest-Adapters.png|Gel image-Adapters File:20170124-qPCR-SeqVsAdap-RCA.png|qPCR Curve </gallery> ===Results-Cycle Threshold=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#4BACC6;font-size:12pt;font-weight:bold" align="center" | width="210" height="37" | Sample | width="85" | Lanes | width="85" | CT Avg | width="85" | CT Diff to PreRCA | width="83" | Amplification | width="85" | CT Diff to 1 amol | width="81" | Amplification to 1 amol |- style="font-size:12pt" | height="15" valign="bottom" | 1 pmol PreRCA | align="center" valign="bottom" | A1,A2 | align="center" align="center" | 17.6 | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" | 1.2 | align="center" align="center" | 2.3 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10 fmol PreRCA | align="center" valign="bottom" | A3,A4 | align="center" align="center" | 19.1 | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" | -0.1 | align="center" align="center" | 0.8 |- style="font-size:12pt" | height="15" valign="bottom" | 100 amol PreRCA | align="center" valign="bottom" | A5,A6 | align="center" align="center" | 18.9 | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" | -0.2 | align="center" align="center" | 1.0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 1 amol PreRCA | align="center" valign="bottom" | A7,A8 | align="center" align="center" | 18.9 | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" | 0.0 | align="center" align="center" | 1.0 |- style="font-size:12pt" | height="15" valign="bottom" | 1 pmol PostRCA | align="center" valign="bottom" | H1,H2 | align="center" align="center" | 11.4 | align="center" align="center" valign="bottom" | 6.25 | align="center" align="center" valign="bottom" | 75.8 | align="center" align="center" | 6.9 | align="center" align="center" | 119.8 |- style="background-color:#DA9694;font-size:12pt" | height="15" valign="bottom" | 10 fmol PostRCA | align="center" valign="bottom" | H3,H4 | align="center" align="center" | 16.3 | align="center" align="center" valign="bottom" | 2.78 | align="center" align="center" valign="bottom" | 6.8 | align="center" align="center" | 2.0 | align="center" align="center" | 3.9 |- style="font-size:12pt" | height="15" valign="bottom" | 100 amol PostRCA | align="center" valign="bottom" | H5,H6 | align="center" align="center" | 18.2 | align="center" align="center" valign="bottom" | 0.69 | align="center" align="center" valign="bottom" | 1.6 | align="center" align="center" | 0.1 | align="center" align="center" | 1.1 |- style="background-color:#DA9694;font-size:12pt" | height="15" valign="bottom" | 1 amol PostRCA | align="center" valign="bottom" | H7,H8 | align="center" align="center" | 18.3 | align="center" align="center" valign="bottom" | 0.56 | align="center" align="center" valign="bottom" | 1.5 | align="center" align="center" | 0.0 | align="center" align="center" | 1.0 |- style="font-size:12pt" | height="15" valign="bottom" | 1 pmol PreRCA Adapters | align="center" | C1,C2 | align="center" align="center" | 16.1 | align="center" | NA | align="center" | NA | align="center" align="center" | 5.1 | align="center" align="center" | 33.4 |- style="background-color:#9BBB59;font-size:12pt" | height="15" valign="bottom" | 1 amol PreRCA Adapters | align="center" | C3,C4 | align="center" align="center" | 21.1 | align="center" | NA | align="center" | NA | align="center" align="center" | 0.0 | align="center" align="center" | 1.0 |- style="font-size:12pt" | height="15" valign="bottom" | 1 pmol PostRCA Adapters | align="center" | C5,C6 | align="center" align="center" | 11.3 | align="center" align="center" | 4.8 | align="center" align="center" | 27.5 | align="center" align="center" | 9.8 | align="center" align="center" | 916.5 |- style="background-color:#9BBB59;font-size:12pt" | height="15" valign="bottom" | 1 amol PostRCA Adapters | align="center" | C7,C8 | align="center" align="center" | 20.8 | align="center" align="center" | 0.4 | align="center" align="center" | 1.3 | align="center" align="center" | 0.4 | align="center" align="center" | 1.3 |} [[Category:ComboLock]] [[Category:20170123]]
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information