DNA extraction High #161904

DNA extraction: High Salt protocol (mosquito)
Protocol
•
•
•
•
Put one fly/wasp in a 1.5 ml eppendorf vial.
Add 50 microliter digestionbuffer
Grind with a sealed yellow pipette tip or a special grinder.
Add 50 microliter digestionbuffer containing 0.2 mg/ml Proteinase K (add the
protK immediately before use, otherwise it will degrade itself!). The standard pK
solution contains 10 mg/ml, so 2 _l is needed per 100 _l of end-volume digestion
buffer.
•
Incubate 2 hrs (at least) at 55 °C
•
•
•
Add 0,4 x volume(= 40 _l) 6 M NaCl
Add 1 x volume (=147 _l) chloroform (in the fume hood) and close the tube firmly.
Mix at least 20 min, on the shake-table (speed ± 70).
•
Centrifuge for 5 min at full speed
•
•
Transfer the water phase (= upper layer) in a new tube (±140 _l). Better leave
some water phase than transferring the white (chloroform) phase.
Add 140 _l iso-propanol and put this for 5 minutes on the shake-table (speed ±
70) at room temperature.
Spin in an eppendorf centrifuge for 15 min.
•
•
•
CAREFULLY remove the supernatant. (The DNA should form a visible pellet)
Add 150 _l 70% ethanol
Spin in an eppendorf centrifuge for 5 min.
•
•
Carefully remove the supernatant
Dry the pellet (1-2 min in the vacuumdryer, medium rate) and dissolve in 20 _l
distilled and autoclaved water. This is your stock DNA
Dilute the stock DNA 10x for the work solution.
•
•
Solutions
6 M NaCl :(Natriumchloride p.a., Merck 1.060404.1000)
350 g per liter MilliQ water (is een verzadigde oplossing)
(bewaren op kamertemperatuur)
Digestionbuffer. (100mM NaCl, 10 mM Tris:HCl pH 8.0, 25 mM EDTA)
Fill a bottle with 80 ml distilled water. Add 2 ml NaCl (5 M), 1 ml Tris:HCl pH
8.0 (1M) and 5 ml EDTA pH 8.0 (0.5 M). Fill up to 97.5 ml and autoclave. After
autoclaving add 2.5 ml SDS (20%)