Genomic DNA from Procyclic T. brucei
DNA Zol Method:
(Use 1ml DNA Zol for each 107 – 108 cells.)
· Pellet cells (0.2 – 1 x 108 cells) 1000 rpm 5’.
· Wash twice with PBS.
· Add 1 ml DNA Zol and pipette up and down slowly 1-2 times.
· Add 0.5 ml 100% rm temp EtOH and invert 2 – 3 times.
Room Temp 1-3 min.
· Pellet in microfuge, 15 min @ 4000 rpm (~1000 x g).
· Wash 1 time in 70% EtOH.
---If necessary, store under 500l 70% EtOH @ 4 deg C.
· Aspirate 70% ethanol and air dry for ~15 min.
§ Dissolve in 100l 8 mM NaOH.
§ Leave at room temp for 1-3 hours if necessary, to completely dissolve DNA.
§ Neutralize with 0.1 M HEPES free acid (18 ml per 100 ml of NaOH).
§ Spin to remove debris, and check the OD260/280.
5 l into 200 l ( = 40X). OD260 x 50 x 40 = mg/ml
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OD260 |
OD280 |
260/280 |
[DNA], mg/ml |
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