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>Djacobse (Created page with "=Circularization Test (Started Daniel:Notebook/ComboLock/2017-1-23|Yesterday]])= Back to Calendar= ==Protocol-Part 2== <ol start="3"> <li>Exon...") |
>Djacobse |
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(15 intermediate revisions by the same user not shown) | |||
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=Circularization Test (Started Daniel:Notebook/ComboLock/2017-1-23|Yesterday]])= | =Circularization Test (Started [[Daniel:Notebook/ComboLock/2017-1-23|Yesterday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
==Protocol-Part 2== | ==Protocol-Part 2== | ||
Line 65: | Line 65: | ||
<li>Incubate at 37C for 3 hours</li> | <li>Incubate at 37C for 3 hours</li> | ||
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> | <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]] |
Latest revision as of 19:01, 16 February 2017
Circularization Test (Started Yesterday)[edit]
Protocol-Part 2[edit]
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare 7X Master mix according to table below
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- qPCR
- Make qPCR master mix according to following recipes
- Add 47 (MM1) or 48 (MM2) uL master mix to each well
- Add 2 uL sample and/or 1 uL appropriate Reverse Primer (1 uM) according to table in following section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 80 uL TBE, 20 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Reagent | uL Added | Master Mix (8.5X) |
Template | 5 | NA |
RCA Primer (10 uM) | 2.5 | 21.25 |
dNTP (1 mM) | 0.8 | 6.8 |
10X Buffer | 2 | 17 |
Phi29 | 1 | 8.5 |
nfH2O | 8.7 | 73.95 |
Total | 20 | 127.5 |
Protocol-Part 3[edit]
Master Mix | Primer Pair | uL Forward Primer | uL Reverse Primer | uL nfH2O | uL 2X SYBR Fast MM | uL Total |
MM1 | AmpF/AmpR | 17.2 | None | 361.2 | 430 | 808.4 |
MM2 | primer6RC/AmpR.Ind29 | 9.2 | 9.2 | 193.2 | 230 | 441.6 |
qPCR Sample Table[edit]
Sample | Product Amt | Pre/Post RCA | Primer Pair | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) | Master Mix |
Sample 0A | 1pmol | Pre | AmpF/AmpR | A1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0B | 1pmol | Pre | AmpF/AmpR | A2 | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2A | 10 fmol | Pre | AmpF/AmpR | A3 | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2B | 10 fmol | Pre | AmpF/AmpR | A4 | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4A | 100 amol | Pre | AmpF/AmpR | A5 | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4B | 100 amol | Pre | AmpF/AmpR | A6 | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6A | 1 amol | Pre | AmpF/AmpR | A7 | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6B | 1 amol | Pre | AmpF/AmpR | A8 | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0AX | 1pmol | Post | AmpF/AmpR | H1 | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0BX | 1pmol | Post | AmpF/AmpR | H2 | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2AX | 10 fmol | Post | AmpF/AmpR | H3 | 26 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2BX | 10 fmol | Post | AmpF/AmpR | H4 | 26 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4AX | 100 amol | Post | AmpF/AmpR | H5 | 27 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4BX | 100 amol | Post | AmpF/AmpR | H6 | 27 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6AX | 1 amol | Post | AmpF/AmpR | H7 | 28 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6BX | 1 amol | Post | AmpF/AmpR | H8 | 28 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0A | 1pmol | Pre | primer6RC/AmpR | C1 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 0B | 1pmol | Pre | primer6RC/AmpR | C2 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6A | 1 amol | Pre | primer6RC/AmpR | C3 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6B | 1 amol | Pre | primer6RC/AmpR | C4 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 0AX | 1pmol | Post | primer6RC/AmpR | C5 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 0BX | 1pmol | Post | primer6RC/AmpR | C6 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6AX | 1 amol | Post | primer6RC/AmpR | C7 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6BX | 1 amol | Post | primer6RC/AmpR | C8 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
NTC | qPCR Primers Only | NA | AmpF/AmpR | F1 | 30 | 2 | 25 | 1 | 1 | 23 | 50 | MM1 |
NTC | qPCR Primers Only | NA | primer6RC/AmpR | F2 | 30 | 2 | 25 | 1 | 1 | 23 | 50 | MM2 |
Results-Curves and Gel Images[edit]
- 20170124-qPCR-PrePostRCA.png
qPCR Curve
- 2017-01-24-RCATest-PreRCA.png
Gel image-Pre RCA
- 2017-01-24-RCATest-PostRCA.png
Gel image-Post RCA
- 2017-01-24-RCATest-Adapters.png
Gel image-Adapters
- 20170124-qPCR-SeqVsAdap-RCA.png
qPCR Curve
Results-Cycle Threshold[edit]
Sample | Lanes | CT Avg | CT Diff to PreRCA | Amplification | CT Diff to 1 amol | Amplification to 1 amol |
1 pmol PreRCA | A1,A2 | 17.6 | NA | NA | 1.2 | 2.3 |
10 fmol PreRCA | A3,A4 | 19.1 | NA | NA | -0.1 | 0.8 |
100 amol PreRCA | A5,A6 | 18.9 | NA | NA | -0.2 | 1.0 |
1 amol PreRCA | A7,A8 | 18.9 | NA | NA | 0.0 | 1.0 |
1 pmol PostRCA | H1,H2 | 11.4 | 6.25 | 75.8 | 6.9 | 119.8 |
10 fmol PostRCA | H3,H4 | 16.3 | 2.78 | 6.8 | 2.0 | 3.9 |
100 amol PostRCA | H5,H6 | 18.2 | 0.69 | 1.6 | 0.1 | 1.1 |
1 amol PostRCA | H7,H8 | 18.3 | 0.56 | 1.5 | 0.0 | 1.0 |
1 pmol PreRCA Adapters | C1,C2 | 16.1 | NA | NA | 5.1 | 33.4 |
1 amol PreRCA Adapters | C3,C4 | 21.1 | NA | NA | 0.0 | 1.0 |
1 pmol PostRCA Adapters | C5,C6 | 11.3 | 4.8 | 27.5 | 9.8 | 916.5 |
1 amol PostRCA Adapters | C7,C8 | 20.8 | 0.4 | 1.3 | 0.4 | 1.3 |