DNA extraction for PacBio
Continued phenol-chloroform extraction using the Thermo Scientific Phenol/Chloroform Extraction and Ethanol Precipitation protocol.
- Added 10 ml Phenol and 10 ml chloroform to sample
- Transferred to glass tube and centrifuged 5 min in Ultracentrigure (in basement)
- Transferred 10 ml of aqueous phase to new tube. Added 10 ml chloroform.
- Centrifuged 5 min in glass tube in Ultracentrifuge
- Transferred about 8.5 ml aqueous phase to new tube. Added 850ul 3M sodium acetate
- Added 25 ml isopropanol. Saw lots of DNA precipitate!
- Incubated 2 hrs at -20°C
- Centrifuged 10 min in glass tube in Ultracentrifuge
At this point, very discouraged because I didn’t see a pellet in bottom of tube. Then remembered that because bottom of tube is rounded, Brian thinks I wouldn’t get a pellet but a thin uniform layer. A light brownish hue around bottom indicated this might be true…
- Eluted in 2000 ul H2O and transferred 500 ul to each of 4 1.5ml microcentrifuge tubes
- Added 750 ul isopropanol to each tube, mixed and centrifuged for 10 min
- Success!!! Nice pellet in bottom of each tube. Eluted each in 20 ul for a total of 80 ul.
Nanodrop: 145 ng/ul in 80 ul = 11.6 ug DNA!
Need fragment analyzer to determine if DNA fragment size is adequate.
Comments from co-authors