CTAB genomic DNA extraction
CTAB extraction buffer (50ml):
5ml Tris 1M, pH 8.0
2ml 0.5M EDTA
add ddH20 to 50ml, add stir bar, spin over low/med heat to dissolve, good on the benchtop for 1-2 weeks
First turn on the waterbath to 60 C and get some liquid nitrogen
- For each extraction, use 500ul CTAB+1ul beta-mercaptoethanol (BME). (make a master mix in a Falcon tube)
- Place ca. 50mg of fresh young leaf tissue in a 1.5ml eppendorf tube, freeze with liquid N2, grind with blue plastic pestle into a fine powder
- Add 500ul of CTAB+BME, grind a little more to thoroughly homogenize tissue and buffer
- Incubate 20min at 60-65 C, inverting or lightly vortexing every 5minutes to keep tissue suspended in buffer
- Spin at 10,000rpm for 2min to pellet cellular debris. Transfer supernatant to new tube
- Add 400ul 24:1 Chloroform: Isoamyl Alcohol and mix by inverting continuously for 2 min (by hand) to precipitate proteins and gunk.
- Spin at 10,000rpm for 5min to separate layers and remove 300ul of top (aqueous) layer to a new tube (being very careful not to touch the bottom layer or the precipitate between the two, which can contaminate/inhibit PCR)
- Add 300ul of isopropanol and invert until DNA precipitates (may take a while so keep rocking until you see strands or at least bubbles, can also put in -20 for 2hrs to overnight to increase precipitation but beware because additional things may also precipitate over long periods).
- Spin at 10,000rpm for 3min to pellet DNA; pour off supernatant
- Add 200ul 70%ETOH, wash pellet by flicking several times, spin again
- Carefully decant 70% (being sure not to pour out pellet) and allow to air dry (overnight in hood or 10min in oven at 50C). You will know it is dry if you flick the tube and don't see any liquid splatter.
- Elute pellet in 50-100ul of water or TE and allow to dissolve a few hours to overnight in 4C fridge. Usually dilute 1:10 or 1:100 for PCR