Daniel:Notebook/ComboLock/2016-11-2
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Circularization Optimization Testing (Started Monday)[edit]
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]
- Size Select Gel
- Mix 10 uL sample (each replicate AB) and 4 uL 6x loading dye into separate tubes for each sample
- Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
- Aliquot 24 uL per sample/ladder lane into each well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- Image in gel doc
- 2016-11-02-CircularizationTest-PostRCA-SizeSelect.png
Before image
- 2016-11-02-CircularizationTest-PostRCA-SizeSelect-After.png
After image
- Centrifuge at 12000 rpm for 1.5 minutes
- Add 500 uL TE to each sample
- Incubate 2 hours at 37C with vigorous shaking;
- Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
- Extract supernatant and add to nanosep column
- Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
- Ethanol Precipitation
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
- Incubate at -80C overnight; continued tomorrow