Daniel:Notebook/ComboLock/2016-7-29: Difference between revisions
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>Djacobse (Created page with "=Phusion Test (Started yesterday)= Back to Calendar ==Protocol== <ol start="3"> <li>Circularization</l...") |
>Djacobse |
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| width="87" | Stock Concentration | | width="87" | Stock Concentration | ||
| width="65" | Reagent Added | | width="65" | Reagent Added | ||
| width=" | | width="105" | uL Added per sample | ||
| width=" | | width="91" | Master Mix (5.1X) | ||
|- style="font-size:12pt" valign="bottom" | |- style="font-size:12pt" valign="bottom" | ||
Line 47: | Line 47: | ||
<ol type="A"> | <ol type="A"> | ||
<li>Set up reaction according to table</li> | <li>Set up reaction according to table</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | |||
| width="200" height="45" | Sample | |||
| width="65" | Lane | |||
| width="65" | AmpR Index | |||
| width="65" | Cells | |||
| width="65" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 100 uM AmpF 6.4 | |||
| width="65" | 10 uM AmpR6.3-IndXX | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 1 (Hs-Hemo Klentaq) | |||
| align="center" valign="bottom" | A1 | |||
| align="center" align="center" valign="bottom" | 15 | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 2 (Hs-Phusion) | |||
| align="center" valign="bottom" | B1 | |||
| align="center" align="center" valign="bottom" | 16 | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 3 (Hs-No C-Probe) | |||
| align="center" valign="bottom" | C1 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 4 (mm-Phusion) | |||
| align="center" valign="bottom" | D1 | |||
| align="center" align="center" valign="bottom" | 18 | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 5 (NTC) | |||
| align="center" valign="bottom" | E1 | |||
| align="center" align="center" valign="bottom" | 19 | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 6 (No Sample) | |||
| align="center" valign="bottom" | F1 | |||
| align="center" align="center" valign="bottom" | 20 | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 19 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|} | |||
<li>Master Mix</li> | |||
<ol type="a"> | |||
<li>152.5 uL KAPA SyberFast MM</li> | |||
<li>103.7 uL nfH2O</li> | |||
<li>3.05 uL AmpF 6.4 100 uM</li></ol> | |||
<li>Add 43 uL Master Mix to each well</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 x20</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol> | |||
</ol> | |||
===qPCR Results=== | |||
<gallery perrow=2 heights=200px widths=200px> | |||
File:2016-07-29-qPCR-PhusionTest.png|qPCR Curve | |||
File:2016-07-29-PhusionTest.png|Gel Image | |||
File:2016-07-29-PhusionTest-RawSample.png|Raw Sample Gel Image (TBU) | |||
File:2016-07-29-PhusionTest-RawSample-Capture2.png|Raw Sample Gel Image (TBU) | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20160728]] |
Latest revision as of 00:15, 18 August 2016
Phusion Test (Started yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Chill on ice 1 min
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 1 uL exonuclease I (20 units/μL) and 1 uL exonuclease III (100 units/μL) per sample
- 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
- Set up reaction according to table
- Master Mix
- 152.5 uL KAPA SyberFast MM
- 103.7 uL nfH2O
- 3.05 uL AmpF 6.4 100 uM
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- 2016-07-29-qPCR-PhusionTest.png
qPCR Curve
- 2016-07-29-PhusionTest.png
Gel Image
- 2016-07-29-PhusionTest-RawSample.png
Raw Sample Gel Image (TBU)
- 2016-07-29-PhusionTest-RawSample-Capture2.png
Raw Sample Gel Image (TBU)
Reagent | Stock Concentration | Reagent Added | uL Added per sample | Master Mix (5.1X) |
ExoI | 20U/uL | 20 | 1 | 5.1 |
ExoIII | 100 U/uL | 100 | 1 | 5.1 |
Sample | Lane | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 (Hs-Hemo Klentaq) | A1 | 15 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 2 (Hs-Phusion) | B1 | 16 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 3 (Hs-No C-Probe) | C1 | 17 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 4 (mm-Phusion) | D1 | 18 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 5 (NTC) | E1 | 19 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 6 (No Sample) | F1 | 20 | 2.5 | 25 | 0.5 | 5 | 19 | 50 |