Daniel:Notebook/ComboLock/2016-12-16: Difference between revisions
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>Djacobse |
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(6 intermediate revisions by the same user not shown) | |||
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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| 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" | |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="160" height="30" | Reagent | ||
| width=" | | width="85" | uL Added | ||
| width=" | | width="85" | Master Mix (13X) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
Line 105: | Line 105: | ||
| height="15" valign="bottom" | RCA Primer (100 uM) | | height="15" valign="bottom" | RCA Primer (100 uM) | ||
| align="center" align="center" valign="bottom" | 2.5 | | align="center" align="center" valign="bottom" | 2.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 32.5 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | dNTP (1 mM) | | height="15" valign="bottom" | dNTP (1 mM) | ||
| align="center" align="center" valign="bottom" | 0.8 | | align="center" align="center" valign="bottom" | 0.8 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 10.4 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | 10X Buffer | | height="15" valign="bottom" | 10X Buffer | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 26 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Phi29 | | height="15" valign="bottom" | Phi29 | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 13 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" align="center" valign="bottom" | 8.7 | | align="center" align="center" valign="bottom" | 8.7 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 113.1 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Total | | 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" | 20 | ||
| align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 195 | ||
|} | |} | ||
Line 335: | Line 335: | ||
</ol> | </ol> | ||
= | ===Results=== | ||
<gallery perrow=2 heights=300px widths=300px> | |||
File:|qPCR curve | |||
File:2016-12-16-MirrorCProbe-ONCap-Supernatant.png|Gel image-supernatant RCA | |||
File:2016-12-16-MirrorCProbe-ONCap-Beads.png|Gel image-Beads RCA | |||
</gallery> | |||
== | ==Adapter Testing== | ||
Just like [[Daniel:Notebook/ComboLock/2016-12-15|yesterday]] I'm going to test presence of C probes and circular product in the pre-RCA mix. | |||
<ol start="7"> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make master mix according to following recipes</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:# | |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="85" height="45" | Master Mix | ||
| width=" | | width="85" | Primers | ||
| width="65" | Primer Target | |||
| width="85" | 2X Kapa SYBR qPCR MM | |||
| width="85" | 10 uM Forward Primer | |||
| width="85" | 10 uM Reverse Primer | |||
| width="65" | H2O | |||
| width="65" | Master Mix | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" | |style="font-weight:bold" height="15" | MMC1 | ||
| align="center" | | align="center" | p24/p4* | ||
| align="center" | C1 | |||
| align="center" align="center" | 230 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 193.2 | |||
| align="center" | MMC1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" | |style="font-weight:bold" height="15" | MMC2 | ||
| align="center" | | align="center" | p24/p4* | ||
| align="center" | C1 | |||
| align="center" align="center" | 230 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 193.2 | |||
| align="center" | MMC1 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" | |style="font-weight:bold" height="15" | MMCP | ||
| align="center" | | align="center" | p6/p12* | ||
| align="center" | C2 | |||
| align="center" align="center" | 230 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 193.2 | |||
| align="center" | MMC2 | |||
|} | |||
<li>Add 48 uL master mix to each well</li> | |||
<li>Add 2 uL sample according to table in the 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 144 uL TBE, 36 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:|qPCR curve displaying sample vs C probes (for supernatant) | |||
File:|qPCR curve displaying sample vs C probes (for beads) | |||
File:|qPCR curve displaying supernatant vs. beads effect | |||
File:2016-12-16-MirrorCProbe-ONCap-Adaptors-CProbes.png|Gel image 1-Sample C probe detection | |||
File:2016-12-16-MirrorCProbe-ONCap-Adaptors-Controls.png|Gel image 2-Controls gel | |||
</gallery> | |||
===qPCR Sample Matrix=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:# | |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="100" height="45" | Sample | ||
| width=" | | width="120" | Condition | ||
| width="91" | | | width="90" | Primers | ||
| width="65" | uL | | width="130" | Primer Target | ||
| width="91" | Fraction | |||
| width="65" | Lane | |||
| width="65" | Sample Vol (uL) | |||
| width="73" | 2X Kapa SYBR qPCR MM | |||
| width="80" | 10 uM Forward Primer | |||
| width="80" | 10 uM Reverse Primer | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
| width="65" | Master Mix | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="30" | |style="font-weight:bold" height="30" | Sample 1A | ||
| align="center" | | | align="center" | Sample | ||
| align="center" | | | align="center" | p24/p4* | ||
| align="center" | C1 | |||
| align="center" | Supernatant-PreRCA | |||
| align="center" | A1 | |||
| align="center" align="center" | 2 | | 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" | MMC1 | |||
|- style="background-color:# | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height=" | |style="font-weight:bold" height="30" | Sample 1B | ||
| align="center" | | | align="center" | Sample | ||
| align="center" | | | align="center" | p24/p4* | ||
| align="center" | C1 | |||
| align="center" | Supernatant-PreRCA | |||
| align="center" | A2 | |||
| align="center" align="center" | 2 | | 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" | MMC1 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height=" | |style="font-weight:bold" height="30" | Sample 1A | ||
| align="center" | | | align="center" | Sample | ||
| align="center" | | | align="center" | p6/p12* | ||
| align="center" align="center" | | | align="center" | C2 | ||
| align="center" | Supernatant-PreRCA | |||
| 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 | |||
| align="center" | MMC2 | |||
|- style="background-color:# | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height=" | |style="font-weight:bold" height="30" | Sample 1B | ||
| align="center" | | | align="center" | Sample | ||
| align="center" | | | align="center" | p6/p12* | ||
| align="center" | C2 | |||
| align="center" | Supernatant-PreRCA | |||
| 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" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
| align="center" | MMC2 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height=" | |style="font-weight:bold" height="30" | Sample 1A | ||
| align="center" | | | align="center" | Sample | ||
| align="center" | | | align="center" | p24/p12* | ||
| align="center" align="center" | | | align="center" | Circular Product | ||
| align="center" | Supernatant-PreRCA | |||
| 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 | |||
| align="center" | MMCP | |||
|- style="background-color:# | |- style="background-color:#BFBFBF;font-size:12pt" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="30" | Sample 1B | ||
| align="center" align="center" | | | align="center" | Sample | ||
| align="center" align="center" | | | align="center" | p24/p12* | ||
| | | align="center" | Circular Product | ||
| align="center" | Supernatant-PreRCA | |||
| 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 | |||
| align="center" | MMCP | |||
| | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" | Sample 1A | |||
| align="center" | Sample | |||
| align="center" | p24/p4* | |||
| align="center" | C1 | |||
| align="center" | Beads-PreRCA | |||
| align="center" | C1 | |||
| 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" | MMC1 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1B | |||
| align="center" | Sample | |||
| align="center" | p24/p4* | |||
| align="center" | C1 | |||
| align="center" | Beads-PreRCA | |||
| align="center" | C2 | |||
| 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" | MMC1 | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height=" | |style="font-weight:bold" height="15" | Sample 1A | ||
| align="center" | | | align="center" | Sample | ||
| align="center" | | | align="center" | p6/p12* | ||
| align="center" | C2 | |||
| align="center" | Beads-PreRCA | |||
| align="center" | C3 | |||
| align="center" align="center" | 2 | | 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" | MMC2 | |||
|- style="background-color:# | |- style="background-color:#DA9694;font-size:12pt" | ||
| height="15" | |style="font-weight:bold" height="15" | Sample 1B | ||
| align="center" | | | align="center" | Sample | ||
| align="center" | | | align="center" | p6/p12* | ||
| align="center" | C2 | |||
| align="center" | Beads-PreRCA | |||
| align="center" | C4 | |||
| align="center" align="center" | 2 | | 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" | MMC2 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" | |style="font-weight:bold" height="15" | Sample 1A | ||
| align="center" | | | align="center" | Sample | ||
| align="center" | | | align="center" | p24/p12* | ||
| align="center" align="center" | | | align="center" | Circular Product | ||
| align="center" | Beads-PreRCA | |||
| align="center" | C5 | |||
| 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" | MMCP | |||
|- style="background-color:# | |- style="background-color:#DA9694;font-size:12pt" | ||
| height="15" | |style="font-weight:bold" height="15" | Sample 1B | ||
| align="center" | | | align="center" | Sample | ||
| align="center" | | | align="center" | p24/p12* | ||
| align="center" | Circular Product | |||
| align="center" | Beads-PreRCA | |||
| align="center" | C6 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
| align="center" | MMCP | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height=" | |style="font-weight:bold" height="30" | Sample 3A | ||
| align="center" | | | align="center" | No C Probes | ||
| align="center" | | | align="center" | p24/p4* | ||
| align="center" align="center" | | | align="center" | C1 | ||
| align="center" | Supernatant-PreRCA | |||
| align="center" | F1 | |||
| 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" | MMC1 | |||
|- style="background-color:# | |- style="background-color:#C4D79B;font-size:12pt" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="30" | Sample 3B | ||
| align="center" align="center" | | | align="center" | No C Probes | ||
| align="center" align="center" | | | align="center" | p24/p4* | ||
| | | align="center" | C1 | ||
| align="center" | Supernatant-PreRCA | |||
| align="center" | F2 | |||
| 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" | MMC1 | |||
| | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="30" | Sample 3A | |||
| align="center" | No C Probes | |||
| align="center" | p6/p12* | |||
| align="center" | C2 | |||
| align="center" | Supernatant-PreRCA | |||
| align="center" | F3 | |||
| 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" | MMC2 | |||
|- style="background-color:#C4D79B;font-size:12pt" | |||
|style="font-weight:bold" height="30" | Sample 3B | |||
| align="center" | No C Probes | |||
| align="center" | p6/p12* | |||
| align="center" | C2 | |||
| align="center" | Supernatant-PreRCA | |||
| align="center" | F4 | |||
| align="center" align="center" | 2 | |||
|- style="background-color:# | | 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" | MMC2 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="30" | |style="font-weight:bold" height="30" | Sample 3A | ||
| align="center" | | | align="center" | No C Probes | ||
| align="center" | | | align="center" | p24/p12* | ||
| align="center" | Circular Product | |||
| align="center" | Supernatant-PreRCA | |||
| align="center" | F5 | |||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
| align="center" align="center" | | 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" | MMCP | |||
|- style="background-color:# | |- style="background-color:#C4D79B;font-size:12pt" | ||
| height=" | |style="font-weight:bold" height="30" | Sample 3B | ||
| align="center" | | | align="center" | No C Probes | ||
| align="center" | | | align="center" | p24/p12* | ||
| align="center" | Circular Product | |||
| align="center" | Supernatant-PreRCA | |||
| align="center" | F6 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
| align="center" align="center" | | align="center" align="center" | 21 | ||
| align="center" align="center" | 50 | |||
| align="center" | MMCP | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height=" | |style="font-weight:bold" height="15" | Sample 3A | ||
| align="center" | | | align="center" | No C Probes | ||
| align="center" | | | align="center" | p24/p4* | ||
| align="center" | C1 | |||
| align="center" | Beads-PreRCA | |||
| align="center" | H1 | |||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
| align="center" align="center" | | 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" | MMC1 | |||
|- style="background-color:# | |- style="background-color:#B1A0C7;font-size:12pt" | ||
| height="15" | |style="font-weight:bold" height="15" | Sample 3B | ||
| align="center" | | | align="center" | No C Probes | ||
| align="center" | | | align="center" | p24/p4* | ||
| align="center" align="center" | | | align="center" | C1 | ||
| align="center" align="center" | | align="center" | Beads-PreRCA | ||
| 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 | |||
| align="center" | MMC1 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" | |style="font-weight:bold" height="15" | Sample 3A | ||
| align="center" align="center" | | | align="center" | No C Probes | ||
| align="center" align="center" | | | align="center" | p6/p12* | ||
| | | align="center" | C2 | ||
| | | align="center" | Beads-PreRCA | ||
| 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 | |||
| align="center" | MMC2 | |||
| | |- style="background-color:#B1A0C7;font-size:12pt" | ||
|style="font-weight:bold" height="15" | Sample 3B | |||
| align="center" | No C Probes | |||
| align="center" | p6/p12* | |||
| align="center" | C2 | |||
| align="center" | Beads-PreRCA | |||
| 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 | |||
| align="center" | MMC2 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3A | |||
| align="center" | No C Probes | |||
| align="center" | p24/p12* | |||
| align="center" | Circular Product | |||
| align="center" | Beads-PreRCA | |||
| 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 | |||
| align="center" | MMCP | |||
|- style="background-color:#B1A0C7;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3B | |||
| align="center" | No C Probes | |||
| align="center" | p24/p12* | |||
| align="center" | Circular Product | |||
| align="center" | Beads-PreRCA | |||
| align="center" | H6 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
|- style="background-color:# | | align="center" align="center" | 1 | ||
| | | align="center" align="center" | 1 | ||
| | | align="center" align="center" | 21 | ||
| | | align="center" align="center" | 50 | ||
| | | align="center" | MMCP | ||
| | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | | |style="font-weight:bold" height="15" valign="bottom" | NTC | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | qPCR Primers Only | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | p24/p4* | ||
| align="center" align="center" valign="bottom" | | | align="center" valign="bottom" | C1 | ||
| align="center" | | align="center" valign="bottom" | NA | ||
| 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" | 23 | |||
| align="center" align="center" | 50 | |||
| align="center" | MMC1 | |||
|- style="background-color:# | |- style="background-color:#FABF8F;font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | | |style="font-weight:bold" height="15" valign="bottom" | NTC | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | qPCR Primers Only | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | p6/p12* | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" valign="bottom" | C2 | ||
| align="center" | | align="center" valign="bottom" | NA | ||
| align="center" | C7 | |||
| 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" | MMC2 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | | |style="font-weight:bold" height="15" valign="bottom" | NTC | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | qPCR Primers Only | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | p24/p12* | ||
| align="center" | | align="center" valign="bottom" | Circular Product | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
| align="center" | | align="center" | F7 | ||
| align="center" | | align="center" align="center" | 2 | ||
| align="center" | | 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" | MMCP | |||
|} | |} | ||
[[Category:ComboLock]] [[Category:20161215]] | [[Category:ComboLock]] [[Category:20161215]] |
Latest revision as of 01:15, 17 December 2016
Overnight Capture (Started Yesterday)[edit]
Protocol[edit]
- Circularization
- Prepare 7X uL Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 60C for 2 hours
- Exonuclease Digestion
- Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour 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; use template from both supernatant only and beads
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- qPCR
- Make master mix according to following recipe
- 151.2 uL nfH2O
- 180 uL SYBR Fast
- 7.2 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table
- 2016-12-16-MirrorCProbe-ONCap-Supernatant.png
Gel image-supernatant RCA
- 2016-12-16-MirrorCProbe-ONCap-Beads.png
Gel image-Beads RCA
- qPCR
- Make master mix according to following recipes
- Add 48 uL master mix to each well
- Add 2 uL sample according to table in the 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 144 uL TBE, 36 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
- 2016-12-16-MirrorCProbe-ONCap-Adaptors-CProbes.png
Gel image 1-Sample C probe detection
- 2016-12-16-MirrorCProbe-ONCap-Adaptors-Controls.png
Gel image 2-Controls gel
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (7x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 56 |
dNTP | 1 mM | 600 pmol | 0.6 | 4.2 |
Betaine | 5 M | 15 umol | 3 | 21 |
10X AmpLigase Buffer | 10X | 1X | 2 | 14 |
Amp Ligase | 5 U/uL | 10 U | 2 | 14 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 22.4 |
nf H2O | 1.2 | 8.4 | ||
Total | 20 | 140 |
Reagent | uL Added | Master Mix (13X) |
Template | 5 | NA |
RCA Primer (100 uM) | 2.5 | 32.5 |
dNTP (1 mM) | 0.8 | 10.4 |
10X Buffer | 2 | 26 |
Phi29 | 1 | 13 |
nfH2O | 8.7 | 113.1 |
Total | 20 | 195 |
Sample | Condition | Fraction | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1A | Sample | Supernatant | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Supernatant | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Supernatant | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Supernatant | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Supernatant | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Supernatant | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | Beads | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Beads | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Beads | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Beads | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Beads | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Beads | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | A7 | 23 | 2 | 25 | 1 | 1 | 23 | 50 |
Results[edit]
Adapter Testing[edit]
Just like yesterday I'm going to test presence of C probes and circular product in the pre-RCA mix.
Master Mix | Primers | Primer Target | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Master Mix |
MMC1 | p24/p4* | C1 | 230 | 9.2 | 9.2 | 193.2 | MMC1 |
MMC2 | p24/p4* | C1 | 230 | 9.2 | 9.2 | 193.2 | MMC1 |
MMCP | p6/p12* | C2 | 230 | 9.2 | 9.2 | 193.2 | MMC2 |
Results[edit]
qPCR Sample Matrix[edit]
Sample | Condition | Primers | Primer Target | Fraction | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) | Master Mix |
Sample 1A | Sample | p24/p4* | C1 | Supernatant-PreRCA | A1 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 1B | Sample | p24/p4* | C1 | Supernatant-PreRCA | A2 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 1A | Sample | p6/p12* | C2 | Supernatant-PreRCA | A3 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 1B | Sample | p6/p12* | C2 | Supernatant-PreRCA | A4 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 1A | Sample | p24/p12* | Circular Product | Supernatant-PreRCA | A5 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 1B | Sample | p24/p12* | Circular Product | Supernatant-PreRCA | A6 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 1A | Sample | p24/p4* | C1 | Beads-PreRCA | C1 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 1B | Sample | p24/p4* | C1 | Beads-PreRCA | C2 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 1A | Sample | p6/p12* | C2 | Beads-PreRCA | C3 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 1B | Sample | p6/p12* | C2 | Beads-PreRCA | C4 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 1A | Sample | p24/p12* | Circular Product | Beads-PreRCA | C5 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 1B | Sample | p24/p12* | Circular Product | Beads-PreRCA | C6 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 3A | No C Probes | p24/p4* | C1 | Supernatant-PreRCA | F1 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 3B | No C Probes | p24/p4* | C1 | Supernatant-PreRCA | F2 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 3A | No C Probes | p6/p12* | C2 | Supernatant-PreRCA | F3 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 3B | No C Probes | p6/p12* | C2 | Supernatant-PreRCA | F4 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 3A | No C Probes | p24/p12* | Circular Product | Supernatant-PreRCA | F5 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 3B | No C Probes | p24/p12* | Circular Product | Supernatant-PreRCA | F6 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 3A | No C Probes | p24/p4* | C1 | Beads-PreRCA | H1 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 3B | No C Probes | p24/p4* | C1 | Beads-PreRCA | H2 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 3A | No C Probes | p6/p12* | C2 | Beads-PreRCA | H3 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 3B | No C Probes | p6/p12* | C2 | Beads-PreRCA | H4 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 3A | No C Probes | p24/p12* | Circular Product | Beads-PreRCA | H5 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 3B | No C Probes | p24/p12* | Circular Product | Beads-PreRCA | H6 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
NTC | qPCR Primers Only | p24/p4* | C1 | NA | A7 | 2 | 25 | 1 | 1 | 23 | 50 | MMC1 |
NTC | qPCR Primers Only | p6/p12* | C2 | NA | C7 | 2 | 25 | 1 | 1 | 23 | 50 | MMC2 |
NTC | qPCR Primers Only | p24/p12* | Circular Product | NA | F7 | 2 | 25 | 1 | 1 | 23 | 50 | MMCP |