Proteinase K method for DNA extraction protocol

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PROTOCOL

1) In 2 ml Eppendorf Safe-Lock tube with precisely upset creature or plant tissues include new 500 μl of extraction cradle (0.8 M guanidine thiocyanate, 10 mM EDTA, 5% Tween 20, 0.5% Triton X-100, 50 mM HEPES-acid*) with 200 μg of proteinase K.


2) vortex quite well and hatch the examples at 55°C for a few hours or better for the time being at 37°-55°C (the more drawn out the better, until disintegrate tissue) with infrequent vortexing. 


3) Include 700 μl of chloroform, vortex very well for 1 moment making an emulsion (in the MM300 Mixer Mill at 30 Hz); alternatively: brood the examples at 55°C during 30 min. 


4) Turn at most extreme speed in a microcentrifuge for 5 minutes. 


5) Move the supernatant into another 2 ml tube containing 500 μl of 2-propanol and 100 μl 3M Na-acetic acid derivation, vortex well overall, and rotator the cylinders at most extreme speed in a microcentrifuge for 4 minutes. 


6) Dispose of the supernatant and include 1.8 ml of 70% ethanol into cylinder and vortex well; rotator the cylinder for 5 minutes at 14000 rpm and again dispose of the supernatant. 


7) Try not to dry DNA pellet and disintegrated quickly in 300 μl of 1xTE, pH 8.0 (with RNAse An) at 55°C for 10-20 minutes.

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