Daniel:Notebook/ComboLock/2016-10-22: Difference between revisions
Jump to navigation
Jump to search
>Djacobse (Created page with "=Cell Test 3 (Started Yesterday)= Back to Calendar ==Protocol== <ol start="3"> <li>Circularization</l...") |
>Djacobse |
||
(11 intermediate revisions by the same user not shown) | |||
Line 127: | Line 127: | ||
<gallery perrow=2 heights=300px widths=300px> | <gallery perrow=2 heights=300px widths=300px> | ||
File:|qPCR Curve | File:20161022-qPCR-CellTest3.png|qPCR Curve | ||
File:|Gel Image | File:2016-10-22-CellTest3.png|Gel Image | ||
</gallery> | </gallery> | ||
==Time Point qPCRs== | |||
Testing the 10 uL aliquots I took [[Daniel:Notebook/ComboLock/2016-10-21|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 [[Daniel:Notebook/ComboLock/2016-10-21|yesterday]]'s and [[Daniel:Notebook/ComboLock/2016-10-21|Thursday]]'s results. | |||
<ol start="8"> | |||
<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="85" height="45" | Master Mix | |||
| width="70" | Forward/Reverse | |||
| width="85" | Forward Primer (uL) | |||
| width="85" | Reverse Primer (uL) | |||
| width="75" | nfH2O (uL) | |||
| width="100" | 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"> | |||
<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="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 | |||
|} | |||
===Results=== | |||
<gallery perrow=4 heights=300px widths=300px> | |||
File:20161022-qPCR-CellTest3-T1.png|qPCR-Time Point 1 | |||
File:20161022-qPCR-CellTest3-T2.png|qPCR-Time Point 2 | |||
File:20161022-qPCR-CellTest3-T3.png|qPCR-Time Point 3 | |||
File:20161022-qPCR-CellTest3-T4.png|qPCR-Time Point 4 | |||
File:2016-10-24-CellTest3-TimePoints-Gel1-Bad.png|Gel-Time Point 1; After C Probes Before Wash | |||
File:2016-10-24-CellTest3-TimePoints-T2.png|Gel-Time Point 2; After C Probes After Wash | |||
File:2016-10-24-CellTest3-TimePoints-T3.png|Gel-Time Point 3; After LP-Hybridization Before Wash | |||
File:2016-10-24-CellTest3-TimePoints-T4.png|Gel-Time Point 4; After LP-Hybridization After Wash | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20161021]] |
Latest revision as of 23:16, 24 October 2016
Cell Test 3 (Started Yesterday)[edit]
Protocol[edit]
- 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[edit]
- 20161022-qPCR-CellTest3.png
qPCR Curve
- 2016-10-22-CellTest3.png
Gel Image
Time Point qPCRs[edit]
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[edit]
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 |
Results[edit]
- 20161022-qPCR-CellTest3-T1.png
qPCR-Time Point 1
- 20161022-qPCR-CellTest3-T2.png
qPCR-Time Point 2
- 20161022-qPCR-CellTest3-T3.png
qPCR-Time Point 3
- 20161022-qPCR-CellTest3-T4.png
qPCR-Time Point 4
- 2016-10-24-CellTest3-TimePoints-Gel1-Bad.png
Gel-Time Point 1; After C Probes Before Wash
- 2016-10-24-CellTest3-TimePoints-T2.png
Gel-Time Point 2; After C Probes After Wash
- 2016-10-24-CellTest3-TimePoints-T3.png
Gel-Time Point 3; After LP-Hybridization Before Wash
- 2016-10-24-CellTest3-TimePoints-T4.png
Gel-Time Point 4; After LP-Hybridization After Wash