Daniel:Notebook/Haplotyping/2016-1-31
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CPT Seq[edit]
High MW DNA Extraction[edit]
- Pellet Cells
- Trypsinize Cells with 0.5mL TrypLE; Incubate 3 min at 37C
- Passage cells if desired
- From the rest, put remainder of cells in 15 mL tube and spin down for 3 min at 1000xg
- Resuspend cells in 200 uL 1X PBS
- Before Starting
- Chill 100% EtOH and 80% EtOH in -20C freezer
- Thaw RNase (if stored at -20C) and keep on ice
- Equilibrate heat blocks or water baths; one to 37C and one to 65C
- Isolation of DNA
- Add 350 uL solution A to cell suspension and vortex in 1 sec intervals until evenly disbursed
- Incubate at 65C for 10 minutes
- Add 150uL Solution B and vortex vigorously until the precipitate moves freely in the tube, and the sample is uniformly viscous (10 sec-1 min)
- Add 500 uL chloroform and vortex until viscosity decreases and the mixture is homogeneous (10 sec-1 min)
- Centrifuge at max speed for 10-20 minutes at 4C to separate phases. Transfer the upper phase into a fresh microcentrifuge tube; recommend 2 mL tube
- DNA Precipitation
- To the DNA solution, add 1 mL of 100% EtOH (-20C) and vortex briefly
- Incubate on ice for 30 minutes
- Centrifuge at max speed for 10-15 minutes at 4C. Remove the ethanol from the pellet with a drawn-out Pasteur pipette
- Add 500 uL of 80% EtOH (-20C) and mix by inverting the tube 3-5 times
- Centrifuge at max speed for 3-5 minutes. Save the pellet and remove the 80% ethanol with drawn out Pasteur pipette
- Centrifuge at max speed for 3-5 minutes at 4C. Remove residual ethanol with a pipettor. Let air dry for 5 minutes.
- Resuspend the pellet in 100 uL TE buffer. Add 2 uL of a 2 mg/mL RNase to bring concentration to 40 ug/mL
- Incubate at 37C for 30 minutes.
- Store at 4 C