Daniel:Notebook/ComboLock/2016-11-2

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Circularization Optimization Testing (Started Monday)[edit]

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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 (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