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=Padlock0301 Dilutions Test (Started [[Daniel:Notebook/ComboLock/2016-10-13|Thursday 10-13]])= I'm going to repeat the PCR test, just in case. <ol start="8"> <li>qPCR</li> <ol type="A"> <li>Prepare the following master mix</li> <ol type="a"> <li>334.25 uL nfH2O</li> <li>9.55 uL 100 uM AmpF-CLv2</li> <li>477.5 uL Kapa SYBR Fast MM</li></ol> <li>Add 43 uL master mix to each well</li> <li>Add sample and AmpR indexes according to sample table in the next 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 176 uL TBE, 44 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="109" height="30" | Sample | width="97" | Template Molecule Count | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="79" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AmpF-CLv2 | width="89" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="77" | Total Volume (uL) |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 6A | 10 amol | A1 | align="center" | 20 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 6B | 10 amol | B1 | align="center" | 20 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 6C | 10 amol | C1 | align="center" | 20 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 8A | 100 zmol | D1 | align="center" | 21 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 8B | 100 zmol | E1 | align="center" | 21 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 8C | 100 zmol | F1 | align="center" | 21 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 9A | 10 zmol | G1 | align="center" | 22 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 9B | 10 zmol | H1 | align="center" | 22 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 9C | 10 zmol | A12 | align="center" | 22 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 10A | 1 zmol | B12 | align="center" | 23 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 10B | 1 zmol | C12 | align="center" | 23 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 10C | 1 zmol | D12 | align="center" | 23 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 11A | 100 ymol | E12 | align="center" | 24 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 11B | 100 ymol | F12 | align="center" | 24 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 11C | 100 ymol | G12 | align="center" | 24 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 12A | 10 ymol | H12 | align="center" | 25 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 12B | 10 ymol | A2 | align="center" | 25 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 12C | 10 ymol | B2 | align="center" | 25 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | NTC | align="center" | 0 | C2 | align="center" | 26 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 19.5 | align="center" | 50 |} ==Results== <gallery perrow=2 heights=300px widths=300px> File:20161017-qPCR-Padlock0301-Dilutions2.png|qPCR image File:2016-10-17-Padlock0301-Dilutions2-Gel1.png|Gel image 1 File:2016-10-17-Padlock0301-Dilutions2-Gel2.png|Gel image 2 </gallery>
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