Daniel:Notebook/ComboLock/2017-3-9: Difference between revisions
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(10 intermediate revisions by the same user not shown) | |||
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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="font-size:12pt" | |- style="font-size:12pt" align="center" valign="center" | ||
| align="center" width=" | | align="center" width="190" height="34" | | ||
|style="background-color:# | |style="background-color:#66CCFF;font-weight:bold" width="85" | Single Rxn | ||
|style="background-color:# | |style="background-color:#66CCFF;font-weight:bold" width="85" | MMC1 (11.2X) | ||
|style="background-color:# | |style="background-color:#66CCFF;font-weight:bold" width="85" | MMC2 (11.2X) | ||
|style="background-color:# | |style="background-color:#66CCFF;font-weight:bold" width="85" | MMP (12.2X) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
Line 37: | Line 37: | ||
| height="15" valign="bottom" | uL Fwd Primer (10 uM) | | height="15" valign="bottom" | uL Fwd Primer (10 uM) | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 11.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 11.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 12.2 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | uL Rev Primer (10 uM) | | height="15" valign="bottom" | uL Rev Primer (10 uM) | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 11.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 11.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 12.2 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | uL nfH2O | | height="15" valign="bottom" | uL nfH2O | ||
| align="center" align="center" valign="bottom" | 21 | | align="center" align="center" valign="bottom" | 21 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 235.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 235.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 256.2 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | uL Kapa SYBR Fast | | height="15" valign="bottom" | uL Kapa SYBR Fast | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 280 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 280 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 305 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Total | | height="15" valign="bottom" | Total | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 48 | |style="font-weight:bold" align="center" align="center" valign="bottom" | 48 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 537.6 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 537.6 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 585.6 | ||
|} | |} | ||
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[[Image:PlateLayout-20170309-BoltTest.png|600px]] | [[Image:PlateLayout-20170309-BoltTest.png|600px]] | ||
*'''Note''': The parentheses in the descriptions indicate which sample was used | |||
*'''Note''':The colors are according to master mix, targeting either C1 (blue), C2 (red), or product (green) | |||
<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 x30</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 360 uL TBE, 90 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 23 minutes at 230V</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> | |||
===Results=== | |||
<gallery perrow=3 heights=300px widths=300px> | |||
File:20170309-qPCR-RFUCurves-BoltTest.png|qPCR curves | |||
File:2017-03-09-BoltTest-C1Target.png|C1 targets | |||
File:2017-03-09-BoltTest-C2Target.png|C2 targets | |||
File:2017-03-09-BoltTest-Product.png|Product targets | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20170308]] | [[Category:ComboLock]] [[Category:20170308]] |
Latest revision as of 22:48, 9 March 2017
Bolt Test (Started Yesterday)[edit]
Protocol[edit]
- Bolt/Latch Hybridization
- Wash twice with 100 uL wash buffer
- Elution
- Add 50 uL wash buffer to each sample
- Cook at 95C for 15 minutes
- qPCR
- Make master mixes according to following recipes
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170309-BoltTest.png
- Note: The parentheses in the descriptions indicate which sample was used
- Note:The colors are according to master mix, targeting either C1 (blue), C2 (red), or product (green)
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 360 uL TBE, 90 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 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Single Rxn | MMC1 (11.2X) | MMC2 (11.2X) | MMP (12.2X) | |
Primer Pair | NA | p24/p4RC | p6/p12RC | p24/p12RC |
uL Fwd Primer (10 uM) | 1 | 11.2 | 11.2 | 12.2 |
uL Rev Primer (10 uM) | 1 | 11.2 | 11.2 | 12.2 |
uL nfH2O | 21 | 235.2 | 235.2 | 256.2 |
uL Kapa SYBR Fast | 25 | 280 | 280 | 305 |
Total | 48 | 537.6 | 537.6 | 585.6 |
Results[edit]
- 20170309-qPCR-RFUCurves-BoltTest.png
qPCR curves
- 2017-03-09-BoltTest-C1Target.png
C1 targets
- 2017-03-09-BoltTest-C2Target.png
C2 targets
- 2017-03-09-BoltTest-Product.png
Product targets