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=Cell Test 3 (Started [[Daniel:Notebook/ComboLock/2016-10-21|Friday 10/21]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] I'm going to try some more qPCR runs in order to delve into this deeper. For one, I'm going to repeat the qPCR from [[Daniel:Notebook/ComboLock/2016-10-22|Saturday]] for time points T1 and T2, since the results are a little strange (I saw nothing in T1 and T2). Second, I'm going to test the adapter based amplifications for latch padlock for several time points, including the last product. ==qPCR Repeats== <ol start="10"> <li>qPCR</li> <ol type="A"> <li>Prepare the following master mixes according to table</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="105" height="45" | Master Mix | width="105" | Forward/Reverse | width="105" | Forward Primer (uL) | width="105" | Reverse Primer (uL) | width="65" | nfH2O (uL) | width="120" | Kapa SYBR Fast MM (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | MM1 | align="center" valign="bottom" | p24RC/p4 | align="center" align="center" valign="bottom" | 0.9 | align="center" align="center" valign="bottom" | 0.9 | align="center" align="center" valign="bottom" | 207.5 | align="center" align="center" valign="bottom" | 227.5 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | MM2 | align="center" valign="bottom" | p6RC/p12 | align="center" align="center" valign="bottom" | 0.9 | align="center" align="center" valign="bottom" | 0.9 | align="center" align="center" valign="bottom" | 207.5 | align="center" align="center" valign="bottom" | 227.5 |} <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample to correct well by following sample table in 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 56 uL TBE, 14 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="105" height="45" | Sample | width="120" | Condition | width="65" | Time Point | width="65" | Lane | width="65" | AmpR-F Combo | width="65" | Sample Vol (uL) | width="105" | 2X Kapa SYBR qPCR MM | width="105" | 100 uM Forward Primer | width="105" | 100 uM Reverse Primer | width="65" | H2O | width="65" | Total Volume (uL) | width="65" | Master Mix |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1 | align="center" | Exp. Rep 1 | align="center" | T1 | align="center" | A1 | align="center" | p24RC/p4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 2 | align="center" | Exp. Rep 2 | align="center" | T1 | align="center" | B1 | align="center" | p24RC/p4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 3 | align="center" | No C Probes | align="center" | T1 | align="center" | C1 | align="center" | p24RC/p4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4 | align="center" | No Cells | align="center" | T1 | align="center" | D1 | align="center" | p24RC/p4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1 | align="center" | Exp. Rep 1 | align="center" | T1 | align="center" | E1 | align="center" | p6RC/p12 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM2 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 2 | align="center" | Exp. Rep 2 | align="center" | T1 | align="center" | F1 | align="center" | p6RC/p12 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 3 | align="center" | No C Probes | align="center" | T1 | align="center" | G1 | align="center" | p6RC/p12 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM2 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4 | align="center" | No Cells | align="center" | T1 | align="center" | H1 | align="center" | p6RC/p12 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1 | align="center" | Exp. Rep 1 | align="center" | T2 | align="center" | A3 | align="center" | p24RC/p4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 2 | align="center" | Exp. Rep 2 | align="center" | T2 | align="center" | B3 | align="center" | p24RC/p4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 3 | align="center" | No C Probes | align="center" | T2 | align="center" | C3 | align="center" | p24RC/p4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4 | align="center" | No Cells | align="center" | T2 | align="center" | D3 | align="center" | p24RC/p4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1 | align="center" | Exp. Rep 1 | align="center" | T2 | align="center" | E3 | align="center" | p6RC/p12 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM2 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 2 | align="center" | Exp. Rep 2 | align="center" | T2 | align="center" | F3 | align="center" | p6RC/p12 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 3 | align="center" | No C Probes | align="center" | T2 | align="center" | G3 | align="center" | p6RC/p12 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM2 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4 | align="center" | No Cells | align="center" | T2 | align="center" | H3 | align="center" | p6RC/p12 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 22.8 | align="center" align="center" | 50 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | NTC | align="center" | No qPCR sample | align="center" | NA | align="center" | A12 | align="center" | p24RC/p4 | align="center" align="center" | 0 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 24.8 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | NTC | align="center" | No qPCR sample | align="center" | NA | align="center" | B12 | align="center" | p6RC/p12 | align="center" align="center" | 0 | align="center" align="center" | 25 | align="center" align="center" | 0.1 | align="center" align="center" | 0.1 | align="center" align="center" | 24.8 | align="center" align="center" | 50 | align="center" | MM2 |} ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20161024-qPCR-CellTest3-T1-Repeat.png|qPCR Results T1; After C Probes Before Wash File:20161024-qPCR-CellTest3-T2-Repeat.png|qPCR Results T2; After C Probes After Wash File:2016-10-24-CellTest3-TimePoints-T1-Repeat.png|Gel Image 1 Repeat; After C Probes Before Wash File:2016-10-24-CellTest3-TimePoints-T2-Repeat.png|Gel Image 2 Repeat; After C Probes After Wash </gallery> ==Additional Analysis== I can test the circular product existence by replacing AmpF with primer12, as [[Daniel:Notebook/ComboLock/2016-10-3|before]]. <ol start="10"> <li>qPCR</li> <ol type="A"> <li>Prepare the following master mix</li> <ol type="a"> <li>107.1 uL nfH2O</li> <li>5.1 uL 10 uM primer 12</li> <li>127.5 uL Kapa SYBR Fast</li></ol> <li>Add 43 uL master mix to each well</li> <li>Add 2 uL sample and 5 uL correct AmpR Index (10 uM) to correct well by following sample 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="105" height="45" | Sample | width="65" | Condition | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="105" | 2X Kapa SYBR qPCR MM | width="105" | 10 uM primer 12 | width="105" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1 | align="center" | Exp. Rep 1 | 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 2 | align="center" | Exp. Rep 2 | align="center" | B1 | 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="30" | Sample 3 | align="center" | No C Probes | align="center" | C1 | 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 4 | align="center" | No Cells | align="center" | D1 | 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="30" valign="bottom" | NTC | align="center" | No qPCR sample | align="center" | E1 | align="center" align="center" | 23 | align="center" align="center" | 0 | 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> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20161024-qPCR-CellTest3-primer12AmpR.png|qPCR Curve File:2016-10-25-CellTest3-p12rc-AmpR.png|Gel Image </gallery> [[Category:ComboLock]] [[Category:20161021]]
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