Daniel:Notebook/ComboLock/2017-1-9
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Version 2 Oligos (Started 1/6/2017)[edit]
From Saturday's results it looks like there is a product at the correct bp size for the version 2 oligos (209bp). What is more exciting is that both sample lanes have it, and none of the negative control lanes have it! So let's send it to the sequencer.
Library Prep[edit]
- Size Select Gel
- Mix 20 uL sample and 4 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)
- 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 225V
- 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
- 2017-01-09-v2OligoSet-SizeSelect.png
Before Image
- 2017-01-09-v2OligoSet-SizeSelect-After.png
After Image
- Centrifuge at 12000 rpm for 1.5 minutes
- Add 500 uL TE to each sample
- Incubate 4 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