Daniel:Notebook/ComboLock/2016-11-2: Difference between revisions

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>Djacobse
(Created page with "=Circularization Optimization Testing (Started Monday)= Back to Calendar Since the product from RCA lo...")
 
>Djacobse
 
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Since the product from RCA looks a little weird, I'm going to run it again and sequence it to see if we got correct product.
Since the product from RCA looks a little weird, I'm going to run it again and sequence it to see if we got correct product.


==Library Prep==
==Library Prep (From [[Daniel:Notebook/ComboLock/2016-11-1|Yesterday]])==


<ol>
<ol>
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<li>Image in gel doc</li>
<li>Image in gel doc</li>
<gallery perrow=2 heights=300px widths=300px>
<gallery perrow=2 heights=300px widths=300px>
File:|Before image
File:2016-11-02-CircularizationTest-PostRCA-SizeSelect.png|Before image
File:|After image
File:2016-11-02-CircularizationTest-PostRCA-SizeSelect-After.png|After image
</gallery>
</gallery>
<li>Centrifuge at 12000 rpm for 1.5 minutes</li>
<li>Centrifuge at 12000 rpm for 1.5 minutes</li>
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<li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2016-11-3|tomorrow]]</li>
<li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2016-11-3|tomorrow]]</li>
</ol></ol>
</ol></ol>
<li>Centrifuge for 25 minutes at 12000 rpm at 4C</li>
<li>Remove supernatant and add 750 uL 70% EtOH</li>
<li>Centrifuge for 12 minutes at 12000 rpm at 4C</li>
<li>Remove supernatant and spin in vacuum centrifuge for 10 minutes</li>
<li>Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A-C by reusing the water</li>




[[Category:ComboLock]] [[Category:20161031]]
[[Category:ComboLock]] [[Category:20161031]]

Latest revision as of 17:44, 3 November 2016

Circularization Optimization Testing (Started Monday)[edit]

Back to Calendar

Since the product from RCA looks a little weird, I'm going to run it again and sequence it to see if we got correct product.

Library Prep (From Yesterday)[edit]

  1. Size Select Gel
    1. Mix 10 uL sample (each replicate AB) and 4 uL 6x loading dye into separate tubes for each sample
    2. Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
    3. Aliquot 24 uL per sample/ladder lane into each well
    4. Run gel for 22 minutes at 250V
    5. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    6. Rinse gel and image in gel doc
    7. Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
    8. Image in gel doc
    9. Centrifuge at 12000 rpm for 1.5 minutes
    10. Add 500 uL TE to each sample
    11. Incubate 2 hours at 37C with vigorous shaking;
    12. Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
    13. Extract supernatant and add to nanosep column
    14. Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
  2. Ethanol Precipitation
    1. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
    2. Incubate at -80C overnight; continued tomorrow