Daniel:Notebook/ComboLock/2016-10-22: Difference between revisions
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>Djacobse |
>Djacobse |
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<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Prepare the following master mix</li> | <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="65" height="45" | Master Mix | |||
| width="56" | Forward/Reverse | |||
| width="65" | Forward Primer (uL) | |||
| width="65" | Reverse Primer (uL) | |||
| width="65" | nfH2O (uL) | |||
| width="65" | 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" | 1.71 | |||
| align="center" align="center" valign="bottom" | 1.71 | |||
| align="center" align="center" valign="bottom" | 389.88 | |||
| align="center" align="center" valign="bottom" | 427.5 | |||
|- 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" | 1.71 | |||
| align="center" align="center" valign="bottom" | 1.71 | |||
| align="center" align="center" valign="bottom" | 389.88 | |||
| align="center" align="center" valign="bottom" | 427.5 | |||
|} | |||
<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"> | <ol type="a"> | ||
<li> | <li>95C 3 min</li> | ||
<li>2 | <li>95C 3 sec</li> | ||
<li> | <li>55C 30 sec</li> | ||
<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="125" | 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="65" | 100 uM Forward Primer | |||
| width="65" | 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="15" | Sample 1 | |||
| align="center" | Exp. Rep 1 | |||
| align="center" | T3 | |||
| align="center" | A6 | |||
| 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:#C4D79B;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2 | |||
| align="center" | Exp. Rep 2 | |||
| align="center" | T3 | |||
| align="center" | B6 | |||
| 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" | T3 | |||
| align="center" | C6 | |||
| 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:#C4D79B;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 4 | |||
| align="center" | No Cells | |||
| align="center" | T3 | |||
| align="center" | D6 | |||
| 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" | T3 | |||
| align="center" | E6 | |||
| 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:#C4D79B;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2 | |||
| align="center" | Exp. Rep 2 | |||
| align="center" | T3 | |||
| align="center" | F6 | |||
| 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" | T3 | |||
| align="center" | G6 | |||
| 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:#C4D79B;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 4 | |||
| align="center" | No Cells | |||
| align="center" | T3 | |||
| align="center" | H6 | |||
| 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" | T4 | |||
| align="center" | A9 | |||
| 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:#B1A0C7;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2 | |||
| align="center" | Exp. Rep 2 | |||
| align="center" | T4 | |||
| align="center" | B9 | |||
| 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" | T4 | |||
| align="center" | C9 | |||
| 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:#B1A0C7;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 4 | |||
| align="center" | No Cells | |||
| align="center" | T4 | |||
| align="center" | D9 | |||
| 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" | T4 | |||
| align="center" | E9 | |||
| 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:#B1A0C7;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2 | |||
| align="center" | Exp. Rep 2 | |||
| align="center" | T4 | |||
| align="center" | F9 | |||
| 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" | T4 | |||
| align="center" | G9 | |||
| 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:#B1A0C7;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 4 | |||
| align="center" | No Cells | |||
| align="center" | T4 | |||
| align="center" | H9 | |||
| 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 | |||
|} |
Revision as of 20:42, 22 October 2016
Cell Test 3 (Started Yesterday)
Protocol
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 7.5 uL exonuclease I (20 units/μL) and 1.5 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Prepare the following master mix
- 89.25 uL nfH2O
- 2.55 uL 100 uM AmpF6.4Sol
- 127.5 uL Kapa SYBR Fast MM
- Add 43 uL master mix to each well
- Add sample and AmpR indexes according to sample table below
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 56 uL TBE, 14 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Condition | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF6.4Sol | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | Exp. Rep 1 | A1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | Exp. Rep 2 | B1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | No C Probes | C1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | No Cells | D1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | No qPCR sample | E1 | 23 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
Results
Time Point qPCRs
Testing the 10 uL aliquots I took yesterday that I took at intervals before/after wash/hybridization steps. I'll be using the direct C-Probe amplification primers, and at a lower concentration than usual after yesterday's and Thursday's results.
- qPCR
- Prepare the following master mixes according to table
- Add 48 uL master mix to each well
- Add 2 uL sample to correct well by following sample table in next section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 56 uL TBE, 14 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Master Mix | Forward/Reverse | Forward Primer (uL) | Reverse Primer (uL) | nfH2O (uL) | Kapa SYBR Fast MM (uL) |
MM1 | p24RC/p4 | 1.71 | 1.71 | 389.88 | 427.5 |
MM2 | p6RC/p12 | 1.71 | 1.71 | 389.88 | 427.5 |
qPCR Sample Table
Sample | Condition | Time Point | Lane | AmpR-F Combo | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM Forward Primer | 100 uM Reverse Primer | H2O | Total Volume (uL) | Master Mix |
Sample 1 | Exp. Rep 1 | T1 | A1 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 2 | Exp. Rep 2 | T1 | B1 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 3 | No C Probes | T1 | C1 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 4 | No Cells | T1 | D1 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 1 | Exp. Rep 1 | T1 | E1 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 2 | Exp. Rep 2 | T1 | F1 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 3 | No C Probes | T1 | G1 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 4 | No Cells | T1 | H1 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 1 | Exp. Rep 1 | T2 | A3 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 2 | Exp. Rep 2 | T2 | B3 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 3 | No C Probes | T2 | C3 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 4 | No Cells | T2 | D3 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 1 | Exp. Rep 1 | T2 | E3 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 2 | Exp. Rep 2 | T2 | F3 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 3 | No C Probes | T2 | G3 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 4 | No Cells | T2 | H3 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 1 | Exp. Rep 1 | T3 | A6 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 2 | Exp. Rep 2 | T3 | B6 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 3 | No C Probes | T3 | C6 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 4 | No Cells | T3 | D6 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 1 | Exp. Rep 1 | T3 | E6 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 2 | Exp. Rep 2 | T3 | F6 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 3 | No C Probes | T3 | G6 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 4 | No Cells | T3 | H6 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 1 | Exp. Rep 1 | T4 | A9 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 2 | Exp. Rep 2 | T4 | B9 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 3 | No C Probes | T4 | C9 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 4 | No Cells | T4 | D9 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 1 | Exp. Rep 1 | T4 | E9 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 2 | Exp. Rep 2 | T4 | F9 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 3 | No C Probes | T4 | G9 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 4 | No Cells | T4 | H9 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
NTC | No qPCR sample | NA | A12 | p24RC/p4 | 0 | 25 | 0.1 | 0.1 | 24.8 | 50 | MM1 |
NTC | No qPCR sample | NA | B12 | p6RC/p12 | 0 | 25 | 0.1 | 0.1 | 24.8 | 50 | MM2 |