Daniel:Notebook/ComboLock/2017-7-17: Difference between revisions

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[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]


==Protocol-Part 4 (Continued from Daniel:Notebook/ComboLock/2017-7-14|Friday July 14]])==
==Protocol-Part 4 (Continued from [[Daniel:Notebook/ComboLock/2017-7-14|Friday July 14]])==
 


<ol start="9">
<li>Bglii digestion</li>
<li>Bglii digestion</li>
<ol type="A">
<ol type="A">
Line 49: Line 49:
<li>Heat kill with 65C for 20 min</li>
<li>Heat kill with 65C for 20 min</li>
<li>Hold at 12C</ol>
<li>Hold at 12C</ol>
<li>qPCR</li>
<ol type="A">
<li>Make 5.2X master mix according to following (1X)</li>
<ol type="a">
<li>109.2 uL nfH<sub>2</sub>O (21)</li>
<li>5.2 uL 10 uM AmpF6.4Sol (1)</li>
<li>130 uL 2X Kapa SYBR Fast Master Mix (25)</li></ol>
<li>Add 47 uL master mix to each well</li>
<li>Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout</li>
[[Image:PlateLayout20170717-MultiplexExperiment.png|250x350px]]
<li>Run the [[Daniel:Protocols/qPCR|Sstandard qPCR protocol]] for ~30 cycles</li>
</ol>
</ol>
<li>TBE Gel</li>
<ol type="A">
<li>Mix together 64 uL TBE and 16 uL 6X dye</li>
<li>Aliquot 10 uL/lane onto parafilm</li>
<li>Add 2 uL correct sample or 1.5 uL 25bp ladder to aliquots</li>
<li>Add 10 uL aliquots to correct lanes</li>
<li>Run gel at 235V for 24 minutes</li>
<li>Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes</li>
<li>Image in gel doc</li></ol>
</ol>
==Results==
<gallery perrow=2 heights=250px widths=350px mode=packed-hover>
File:PlateLayout20170717-MultiplexExperiment.png|Plate layout
File:Multiplex-20170717-plateCTs-normed.png|Plate CTs
File:Multiplex-20170717-rawcurves.png|Raw curves
File:2017-07-17-MultiplexComboLock.png|Gel image
</gallery>
[[Category:ComboLock]] [[Category:20170713]]

Latest revision as of 19:09, 18 July 2017

Multiplex Experiment (Started Thursday July 13)[edit]

Back to Calendar

Protocol-Part 4 (Continued from Friday July 14)[edit]

  1. Bglii digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (4.2X)
      RCA Rxn 7 0
      10X Buffer 3.1 2 8.4
      Bglii 1 0
      nfH2O 10 42
      Total 20 50.4
    3. Aliquot 12 uL master mix into new tube
    4. Add 7 uL appropriate sample
    5. Mix with vortexer, spin down, and add 1 uL Bglii
    6. Incubate 2 hr 37C
    7. Heat kill with 65C for 20 min
    8. Hold at 12C
  2. qPCR
    1. Make 5.2X master mix according to following (1X)
      1. 109.2 uL nfH2O (21)
      2. 5.2 uL 10 uM AmpF6.4Sol (1)
      3. 130 uL 2X Kapa SYBR Fast Master Mix (25)
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout
    4. File:PlateLayout20170717-MultiplexExperiment.png
    5. Run the Sstandard qPCR protocol for ~30 cycles
  3. TBE Gel
    1. Mix together 64 uL TBE and 16 uL 6X dye
    2. Aliquot 10 uL/lane onto parafilm
    3. Add 2 uL correct sample or 1.5 uL 25bp ladder to aliquots
    4. Add 10 uL aliquots to correct lanes
    5. Run gel at 235V for 24 minutes
    6. Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes
    7. Image in gel doc

Results[edit]