Daniel:Notebook/ComboLock/2017-3-26: Difference between revisions

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>Djacobse
(Created page with "=RCA Test= Back to Calendar ==Linear Product Production-Bglii== <ol start="2"> <li>TBE gel</li> <ol type="A"> <li>Mix 64 uL TBE and 16 uL 6X ...")
 
>Djacobse
Line 27: Line 27:
<ol start="3">
<ol start="3">
<li>ssDNA column</li>
<li>ssDNA column</li>
<ol type="A">
<li>Add 50 uL '''Binding Buffer''' to the sample; mix well</li>
<li>Add 50 uL '''Binding Buffer''' to the sample; mix well</li>
<li>Transfer to '''IIC Column''' and centrifuge at 14000 rpm for 1 minute; '''SAVE THE FLOW THROUGH'''</li>
<li>Transfer to '''IIC Column''' and centrifuge at 14000 rpm for 1 minute; '''SAVE THE FLOW THROUGH'''</li>

Revision as of 23:07, 26 March 2017

RCA Test

Back to Calendar


Linear Product Production-Bglii

  1. TBE gel
    1. Mix 64 uL TBE and 16 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample/ladder
    3. Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
    4. File:GelLanes-20170325-LinearProduct.png
    5. Add 10 uL mix to gel lanes
    6. Run gel for 24 minutes at 230V
    7. Stain with 2 uL SYBR gold for 3 minutes
    8. Rinse and image in gel doc

File:2017-03-26-LinearProductProduction-Bglii.png

That's the right one. However, there is a ton of primer (bottom of the gel), so I'll run a ssDNA column to fix.

  1. ssDNA column
    1. Add 50 uL Binding Buffer to the sample; mix well
    2. Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
    3. Add 70 uL 100% EtOH to flow through; mix well
    4. Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
    5. Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    6. Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    7. Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    8. Centrifuge empty column at 14000 rpm for 2 minutes
    9. Transfer to empty 1.5mL centrifuge tube (low bind)
    10. Add 20 uL nfH2O and centrifuge at 14000 rpm for 1 minute