Daniel:Notebook/ComboLock/2016-8-28: Difference between revisions
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>Djacobse (Created page with "=Positive Control Dilution Test (Started Friday)= Back to Calendar ==Protocol== <ol start="2"> <li>Cir...") |
>Djacobse |
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(8 intermediate revisions by the same user not shown) | |||
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<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="105" height="45" | Sample | |||
| width="78" | Molecule Count | |||
| width="65" | Lane | |||
| width="65" | AmpR Index | |||
| width="65" | Sample Vol (uL) | |||
| 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 0 | |||
| align="center" valign="bottom" | 1.00E+11 | |||
| align="center" valign="bottom" | A1 | |||
| align="center" align="center" valign="bottom" | 34 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 1 | |||
| align="center" valign="bottom" | 1.00E+10 | |||
| align="center" valign="bottom" | B1 | |||
| align="center" align="center" valign="bottom" | 35 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 2 | |||
| align="center" valign="bottom" | 1.00E+09 | |||
| align="center" valign="bottom" | C1 | |||
| align="center" align="center" valign="bottom" | 36 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 3 | |||
| align="center" valign="bottom" | 1.00E+08 | |||
| align="center" valign="bottom" | D1 | |||
| align="center" align="center" valign="bottom" | 37 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 4 | |||
| align="center" valign="bottom" | 1.00E+07 | |||
| align="center" valign="bottom" | E1 | |||
| align="center" align="center" valign="bottom" | 38 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 5 | |||
| align="center" valign="bottom" | 1.00E+06 | |||
| align="center" valign="bottom" | F1 | |||
| align="center" align="center" valign="bottom" | 39 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 6 | |||
| align="center" valign="bottom" | 100000 | |||
| align="center" valign="bottom" | G1 | |||
| align="center" align="center" valign="bottom" | 41 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 7 | |||
| align="center" valign="bottom" | 10000 | |||
| align="center" valign="bottom" | H1 | |||
| align="center" align="center" valign="bottom" | 42 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 8 | |||
| align="center" valign="bottom" | 1000 | |||
| align="center" valign="bottom" | A12 | |||
| align="center" align="center" valign="bottom" | 43 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 9 | |||
| align="center" valign="bottom" | 100 | |||
| align="center" valign="bottom" | B12 | |||
| align="center" align="center" valign="bottom" | 44 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 10 | |||
| align="center" valign="bottom" | 10 | |||
| align="center" valign="bottom" | C12 | |||
| align="center" align="center" valign="bottom" | 45 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 11 | |||
| align="center" valign="bottom" | 1 | |||
| align="center" valign="bottom" | D12 | |||
| align="center" align="center" valign="bottom" | 46 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 12 | |||
| align="center" valign="bottom" | 0 | |||
| align="center" valign="bottom" | E12 | |||
| align="center" align="center" valign="bottom" | 47 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| 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" | 19.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|} | |||
<li>Master Mix</li> | <li>Master Mix</li> | ||
Line 35: | Line 206: | ||
<li>plate read</li> | <li>plate read</li> | ||
<li>goto b x20</li> | <li>goto b x20</li> | ||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
<li>qPCR Standard</li> | |||
<ol type="A"> | |||
<li>Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC | |||
<li>Master Mix</li> | |||
<ol type="a"> | |||
<li>352.5 uL KAPA SyberFast MM</li> | |||
<li>310.2 uL nfH2O</li> | |||
<li>7.05 uL primer2</li> | |||
<li>7.05 uL primer12RC</li></ol> | |||
<li>Add 48 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 x25</li> | |||
<li>72C 2 min</li> | <li>72C 2 min</li> | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol> | </ol> | ||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | <gallery perrow=2 heights=300px widths=300px> | ||
File:|X2 qPCR Curve | File:20160828-X2Dilution-qPCR.png|X2 Dilutions qPCR Curve | ||
File:|X2 | File:20160828-X2Dilution-Standard-qPCR.png|X2 Dilutions Standard qPCR Curve | ||
File:2016-08-28-X2Dilution-0-5.png|Samples 0-5 | |||
File:2016-08-28-X2Dilution-6-11.png|Samples 0-5 | |||
File:20160829-X2Dilution-Primers-qPCR.png|Primer Amplification of Sample Amplicons; [[Daniel:Notebook/ComboLock/2016-8-29|08/29/2016]] | |||
</gallery> | </gallery> | ||
[[Category:ComboLock]] [[Category:20160826]] |
Latest revision as of 20:05, 29 August 2016
Positive Control Dilution Test (Started Friday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 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
- Set up reaction according to table
- Master Mix
- 327.5 uL KAPA SyberFast MM
- 229.25 uL nfH2O
- 6.55 uL AmpF 6.4
- 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
- qPCR Standard
- Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC
- Master Mix
- 352.5 uL KAPA SyberFast MM
- 310.2 uL nfH2O
- 7.05 uL primer2
- 7.05 uL primer12RC
- Add 48 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x25
- 72C 2 min
- 16C hold
- 20160828-X2Dilution-qPCR.png
X2 Dilutions qPCR Curve
- 20160828-X2Dilution-Standard-qPCR.png
X2 Dilutions Standard qPCR Curve
- 2016-08-28-X2Dilution-0-5.png
Samples 0-5
- 2016-08-28-X2Dilution-6-11.png
Samples 0-5
- 20160829-X2Dilution-Primers-qPCR.png
Primer Amplification of Sample Amplicons; 08/29/2016
Sample | Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 0 | 1.00E+11 | A1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1 | 1.00E+10 | B1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | 1.00E+09 | C1 | 36 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | 1.00E+08 | D1 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | 1.00E+07 | E1 | 38 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | 1.00E+06 | F1 | 39 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | 100000 | G1 | 41 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | 10000 | H1 | 42 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8 | 1000 | A12 | 43 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9 | 100 | B12 | 44 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10 | 10 | C12 | 45 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 11 | 1 | D12 | 46 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 12 | 0 | E12 | 47 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |