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=Mirror Probe Test (Started [[Daniel:Notebook/ComboLock/2016-12-13|Tuesday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==RCA on Beads/Supernatant Fractions== <ol start="7"> <li>qPCR</li> <ol type="A"> <li>Make master mix according to following recipe</li> <ol type="a"> <li>298.2 uL nfH2O</li> <li>355 uL SYBR Fast</li> <li>14.2 uL 10 uM AmpF</li> </ol> <li>Add 47 uL master mix to each well</li> <li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section</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="90" height="45" | Sample | width="120" | Condition | width="100" | Fraction | width="85" | Lane | width="85" | AmpR Index | width="85" | Sample Vol (uL) | width="105" | 2X Kapa SYBR qPCR MM | width="85" | 10 uM Forward Primer | width="85" | 10 uM Reverse Primer | width="85" | H2O | width="85" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample | align="center" | Supernatant | align="center" | A1 | align="center" align="center" | 20 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | align="center" | Supernatant | align="center" | A2 | align="center" align="center" | 20 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 2A | align="center" | No Template | align="center" | Supernatant | align="center" | A3 | align="center" align="center" | 21 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" valign="bottom" | No Template | align="center" | Supernatant | align="center" | A4 | align="center" align="center" | 21 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3A | align="center" | No C Probe | align="center" | Supernatant | align="center" | A5 | align="center" align="center" | 22 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3B | align="center" | No C Probe | align="center" | Supernatant | align="center" | A6 | align="center" align="center" | 22 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample | align="center" | Beads | align="center" | H1 | align="center" align="center" | 23 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | align="center" | Beads | align="center" | H2 | align="center" align="center" | 23 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 2A | align="center" | No Template | align="center" | Beads | align="center" | H3 | align="center" align="center" | 24 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" valign="bottom" | No Template | align="center" | Beads | align="center" | H4 | align="center" align="center" | 24 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3A | align="center" | No C Probe | align="center" | Beads | align="center" | H5 | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3B | align="center" | No C Probe | align="center" | Beads | align="center" | H6 | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" | height="30" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" valign="bottom" | NA | align="center" | A7 | 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" | 23 | 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 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:20161215-qPCR-MirrorCProbe-PostRCA.png|qPCR curves File:2016-12-15-MirrorCProbe-RCA-Supernatant.png|Gel image-supernatant RCA File:2016-12-15-MirrorCProbe-RCA-Beads.png|Gel image-beads RCA </gallery> Although there is a slight smear in the region of correct size (211bp), the smear is present in every lane (except the NTC). This suggests the smear is a result of the beads in general and not really related to the product. '''My conclusion is that the circular product never formed'''. ==C Probe and Product Detection== I'm now going to investigate the presence/absence of C probes and circularized products using the adapter primers. <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> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="85" height="45" | Master Mix | 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-weight:bold" height="15" | MMC1 | 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="font-weight:bold" height="15" | MMC2 | 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-weight:bold" height="15" | MMCP | 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:20161215-qPCR-MirrorCProbes-Adapters-SamplevsControl-Beads.png|qPCR curve displaying sample vs C probes (for supernatant) File:20161215-qPCR-MirrorCProbes-Adapters-SamplevsControl.png|qPCR curve displaying sample vs C probes (for beads) File:20161215-qPCR-MirrorCProbes-Adapters-SupvsBeads.png|qPCR curve displaying supernatant vs. beads effect File:2016-12-15-MirrorCProbe-Adapters-CProbeDetection.png|Gel image 1-Sample C probe detection File:2016-12-15-MirrorCProbe-Adapters-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> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="100" height="45" | Sample | width="120" | Condition | width="90" | Primers | 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-weight:bold" height="30" | Sample 1A | align="center" | Sample | align="center" | p24/p4* | align="center" | C1 | align="center" | Supernatant-PreRCA | 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 | align="center" | MMC1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="30" | Sample 1B | align="center" | Sample | align="center" | p24/p4* | align="center" | C1 | align="center" | Supernatant-PreRCA | 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 | align="center" | MMC1 |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 1A | align="center" | Sample | align="center" | p6/p12* | 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:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="30" | Sample 1B | align="center" | Sample | 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" | 21 | align="center" align="center" | 50 | align="center" | MMC2 |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 1A | align="center" | Sample | align="center" | p24/p12* | 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:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="30" | Sample 1B | align="center" | Sample | 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-weight:bold" height="15" | Sample 1A | align="center" | Sample | align="center" | p6/p12* | align="center" | C2 | align="center" | Beads-PreRCA | align="center" | C3 | 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:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | align="center" | p6/p12* | align="center" | C2 | align="center" | Beads-PreRCA | align="center" | C4 | 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 1A | align="center" | Sample | align="center" | p24/p12* | 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:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | 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" | 21 | align="center" align="center" | 50 | align="center" | MMCP |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 3A | align="center" | No C Probes | align="center" | p24/p4* | 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:#C4D79B;font-size:12pt" |style="font-weight:bold" height="30" | Sample 3B | align="center" | No C Probes | 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 | 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="30" | Sample 3A | align="center" | No C Probes | align="center" | p24/p12* | align="center" | Circular Product | align="center" | Supernatant-PreRCA | align="center" | F5 | 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:#C4D79B;font-size:12pt" |style="font-weight:bold" height="30" | Sample 3B | align="center" | No C Probes | 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" | 21 | align="center" align="center" | 50 | align="center" | MMCP |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3A | align="center" | No C Probes | align="center" | p24/p4* | align="center" | C1 | align="center" | Beads-PreRCA | 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 | align="center" | MMC1 |- style="background-color:#B1A0C7;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3B | align="center" | No C Probes | align="center" | p24/p4* | align="center" | C1 | 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-weight:bold" height="15" | Sample 3A | align="center" | No C Probes | 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 | 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" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" valign="bottom" | p24/p4* | align="center" valign="bottom" | C1 | 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:#FABF8F;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" valign="bottom" | p6/p12* | align="center" valign="bottom" | C2 | 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-weight:bold" height="15" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" valign="bottom" | p24/p12* | align="center" valign="bottom" | Circular Product | align="center" valign="bottom" | NA | align="center" | F7 | 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" | MMCP |}
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