Daniel:Notebook/ComboLock/2017-3-9: Difference between revisions

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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="200" height="32"  valign="bottom" | &nbsp;
| align="center" width="190" height="34" | &nbsp;
|style="background-color:#8DB4E2;font-weight:bold" width="79" align="center" | Single Rxn
|style="background-color:#66CCFF;font-weight:bold" width="85" | Single Rxn
|style="background-color:#8DB4E2;font-weight:bold" width="65" align="center" | MMC1
|style="background-color:#66CCFF;font-weight:bold" width="85" | MMC1 (11.2X)
|style="background-color:#8DB4E2;font-weight:bold" width="65" align="center" | MMC2
|style="background-color:#66CCFF;font-weight:bold" width="85" | MMC2 (11.2X)
|style="background-color:#8DB4E2;font-weight:bold" width="65" align="center" | MMP
|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" | 10.2
| align="center" align="center" valign="bottom" | 11.2
| align="center" align="center" valign="bottom" | 10.2
| align="center" align="center" valign="bottom" | 11.2
| align="center" align="center" valign="bottom" | 10.2
| 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" | 10.2
| align="center" align="center" valign="bottom" | 11.2
| align="center" align="center" valign="bottom" | 10.2
| align="center" align="center" valign="bottom" | 11.2
| align="center" align="center" valign="bottom" | 10.2
| 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" | 214.2
| align="center" align="center" valign="bottom" | 235.2
| align="center" align="center" valign="bottom" | 214.2
| align="center" align="center" valign="bottom" | 235.2
| align="center" align="center" valign="bottom" | 214.2
| 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" | 255
| align="center" align="center" valign="bottom" | 280
| align="center" align="center" valign="bottom" | 255
| align="center" align="center" valign="bottom" | 280
| align="center" align="center" valign="bottom" | 255
| 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" | 489.6
|style="font-weight:bold" align="center" align="center" valign="bottom" | 537.6
|style="font-weight:bold" align="center" align="center" valign="bottom" | 489.6
|style="font-weight:bold" align="center" align="center" valign="bottom" | 537.6
|style="font-weight:bold" align="center" align="center" valign="bottom" | 489.6
|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]

Back to Calendar

Protocol[edit]

  1. Bolt/Latch Hybridization
    1. Wash twice with 100 uL wash buffer
  2. Elution
    1. Add 50 uL wash buffer to each sample
    2. Cook at 95C for 15 minutes
  3. qPCR
    1. Make master mixes according to following recipes
    2.   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
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. 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)
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  4. TBE Gel
    1. Mix 360 uL TBE, 90 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 23 minutes at 230V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]