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=v3 Oligos-Round 3-Low Temp (Started [[Daniel:Notebook/ComboLock/2017-2-7|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-Part 4== <ol start="9"> <li>qPCR</li> <ol type="A"> <li>Make 17.2X qPCR master mix according to following recipe</li> <ol type="a"> <li>361.2 uL nfH2O</li> <li>430 uL SYBR Fast</li> <li>17.2 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</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="110" height="45" | Sample | width="180" | Condition | width="65" | Pre/Post RCA | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="95" | 2X Kapa SYBR qPCR MM | width="95" | 10 uM Forward Primer | width="95" | 10 uM Reverse Primer | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample | align="center" | Pre | 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" | Pre | 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" | Pre | 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" valign="bottom" | Pre | 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" | Pre | 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" | Pre | 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 4A | align="center" | No Lock Phosphate | align="center" | Pre | align="center" | A7 | 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:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4B | align="center" | No Lock Phosphate | align="center" valign="bottom" | Pre | align="center" | A8 | 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 1AX | align="center" | Sample | align="center" | Post | align="center" | H1 | 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" | Sample 1BX | align="center" | Sample | align="center" | Post | align="center" | H2 | 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 2AX | align="center" | No Template | align="center" | Post | align="center" | H3 | 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" valign="bottom" | Sample 2BX | align="center" valign="bottom" | No Template | align="center" valign="bottom" | Post | align="center" | H4 | 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" |style="font-weight:bold" height="15" | Sample 3AX | align="center" | No C Probe | align="center" | Post | align="center" | H5 | 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 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3BX | align="center" | No C Probe | align="center" | Post | align="center" | H6 | 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 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 4AX | align="center" | No Lock Phosphate | align="center" | Post | align="center" | H7 | 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 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4BX | align="center" | No Lock Phosphate | align="center" valign="bottom" | Post | align="center" | H8 | 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 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" align="center" valign="bottom" | | align="center" | D5 | 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 |} <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 192 uL TBE, 48 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 23 minutes at 230V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ==Results-Normal Protocol== <gallery perrow=2 heights=300px widths=300px> File:20170208-qPCR-v3Oligos-LoTemp.png|qPCR curve-Normal protocol File:2017-02-08-v3Oligos-LoTemp-PreRCA.png|Gel image-Pre RCA File:2017-02-08-v3Oligos-LoTemp-PostRCA.png|Gel image-Post RCA File:2017-02-08-v3Oligos-LoTemp-PostRCA-Highlight.png|Gel image-Post RCA-Product Highlighted </gallery> As the highlighted band shows, sample A might actually have amplified since there is a faint band at the correct size that is not in the other lanes. However, it is faint and about equal to the incorrect band at 200bp. ==Protocol-Part 5== <ol start="8"> <li>Ethanol Precipitation</li> <ol type="A" start="5"> <li>Centrifuge at 4C for 30 minutes at 12000 rpm</li> <li>Remove supernatant and add 750 uL cold 70%EtOH</li> <li>Centrifuge at 4C for 20 minutes at 12000 rpm</li> <li>Remove supernatant and vacuum centrifuge for 15 minutes to completely dry</li> <li>Resuspend pellet in 20 uL nfH2O</li></ol></ol> <ol start="11"> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 8.2X Master mix according to table below; use LLRC primer for '''A''' samples and LLRC* (phosphorothiorate) samples for '''B''' samples</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="190" height="30" | Reagent | width="65" | uL Added | width="85" | Master Mix (8.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" align="center" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 0 |- 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" | 20.5 |- 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" | 6.56 |- 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" | 16.4 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 10.7 | align="center" align="center" valign="bottom" | 87.74 |- 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" | 131.2 |} <li>Incubate at 30C for 6 hours</li> <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li> </ol></ol> ==Protocol-Part 6== <ol start="12"> <li>qPCR</li> <ol type="A"> <li>Make 17.2X qPCR master mix according to following recipe</li> <ol type="a"> <li>361.2 uL nfH2O</li> <li>430 uL SYBR Fast</li> <li>17.2 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</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="120" height="45" | Sample | width="190" | Condition | width="65" | Pre/Post RCA | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="95" | 2X Kapa SYBR qPCR MM | width="95" | 10 uM Forward Primer | width="95" | 10 uM Reverse Primer | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A* | align="center" | Sample-EtOH+ | align="center" | Pre | 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-EtOH+ | align="center" | Pre | 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-EtOH+ | align="center" | Pre | 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-EtOH+ | align="center" valign="bottom" | Pre | 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-EtOH+ | align="center" | Pre | 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-EtOH+ | align="center" | Pre | 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 4A* | align="center" | No Lock Phosphate-EtOH+ | align="center" | Pre | align="center" | A7 | 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:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4B* | align="center" | No Lock Phosphate-EtOH+ | align="center" valign="bottom" | Pre | align="center" | A8 | 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 1AZ | align="center" | Sample-EtOH+ | align="center" | Post | align="center" | H1 | 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" | Sample 1BZ | align="center" | Sample-EtOH+ | align="center" | Post | align="center" | H2 | 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 2AZ | align="center" | No Template-EtOH+ | align="center" | Post | align="center" | H3 | 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" valign="bottom" | Sample 2BZ | align="center" valign="bottom" | No Template-EtOH+ | align="center" valign="bottom" | Post | align="center" | H4 | 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" |style="font-weight:bold" height="15" | Sample 3AZ | align="center" | No C Probe-EtOH+ | align="center" | Post | align="center" | H5 | 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 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3BZ | align="center" | No C Probe-EtOH+ | align="center" | Post | align="center" | H6 | 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 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 4AZ | align="center" | No Lock Phosphate-EtOH+ | align="center" | Post | align="center" | H7 | 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 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4BZ | align="center" | No Lock Phosphate-EtOH+ | align="center" valign="bottom" | Post | align="center" | H8 | 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 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" align="center" valign="bottom" | | align="center" | D5 | 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 |} <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 192 uL TBE, 48 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 23 minutes at 230V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol> <li>TBE Gel-Unamplified EtOH Precipitation product</li> <ol type="A"> <li>Mix 60 uL TBE, 20 uL 6x loading dye</li> <li>Aliquot 8 uL per sample/ladder lane onto parafilm</li> <li>Add 4 uL of sample or 1 uL ladder, 3 uL TBE to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 23 minutes at 230V</li> <li>Open gel and stain with 2 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ==Results-Ethanol Precipitation== <gallery perrow=2 heights=300px widths=300px> File:20170208-qPCR-v3Oligos-LoTemp-EtOH.png|qPCR curve-Normal protocol File:2017-02-08-v3Oligos-LoTemp-PreRCA-EtOH.png|Gel image-Pre RCA File:2017-02-08-v3Oligos-LoTemp-PostRCA-EtOH.png|Gel image-Post RCA File:2017-02-08-v3Oligos-LoTemp-AfterEtOH.png|Purified samples after EtOH before amplification </gallery> [[Category:ComboLock]] [[Category:20170207]]
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