Daniel:Notebook/ComboLock/2016-11-2

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Revision as of 18:23, 2 November 2016 by >Djacobse (→‎Library Prep)
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Circularization Optimization Testing (Started Monday)

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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.

Library Prep

  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