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=Version 3 Oligos (Started [[Daniel:Notebook/ComboLock/2017-1-13|Thursday, Jan 13]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Product Check== Searching for the bridge product (or circular product) using the primer24/primer12 combination. <ol> <li>qPCR</li> <ol type="A"> <li>Make 5.2X qPCR master mix according to following recipe</li> <ol type="a"> <li>109.2 uL nfH2O</li> <li>130 uL SYBR Fast</li> <li>5.2 uL 10 uM primer24RC</li> <li>5.2 uL 10 uM primer24RC</li> </ol> <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample 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="101" | Condition | width="65" | Lane | 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) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample | 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 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | 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 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 4A | align="center" | No Lock Phosphate | 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 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4B | align="center" | No Lock Phosphate | 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 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | 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" | 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 64 uL TBE, 16 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 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=== <gallery perrow=-2 heights=300px widths=300px> File:20170111-qPCR-v3Oligos-Adapters.png|qPCR curves File:2017-01-18-v3Oligos-Adapters.png|Gel image </gallery> ==RCA Repeat== Gonna try RCA again, since the adapter testing above showed product with at least the adapter regions. That means either the bridge is present or the circular product itself. So repeating RCA should increase the concentration of product, if available. <ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 8.5X Master mix according to table below</li> <li>Add 15 uL master mix to each tube</li> <li>Add sample to new reaction tubes according to following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="160" height="30" | Reagent | width="65" | uL Added | width="90" | Master Mix (8.5X) |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer (LinkerL-RevComp; 10 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 21.25 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="center" align="center" valign="bottom" | 0.8 | align="center" align="center" valign="bottom" | 6.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Phi29 Buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 17 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 8.5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 8.7 | align="center" align="center" valign="bottom" | 73.95 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 127.5 |} <li>Incubate at 37C for 3 hours</li> <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> Continued [[Daniel:Notebook/ComboLock/2017-1-19|tomorrow]] [[Category:ComboLock]] [[Category:20170113]]
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