Digestion NotI
Add : - nuclease - free water qsp 50 µL > 47 µL - 5µL 10X Buffer O - 1µL DNA ( 1 µg / µL ) - 1 µL NotI 1 ( 10U / µL ) * .
Mix gently and spin down for a few seconds .
Incubate at 37°C for 1 - 16 hours .
The digestion reaction may be scaled either up or down .
It's possible to process extended incubation by adding 0.25U / µg of DNA in 50 µL of reaction volume .
incubation at 80°C for 20 min .
Measure the volume of the DNA sample > 50 µL
Add 1 / 10 volume of sodium acetate , pH 5.2 , ( final concentration of 0.3 M )
- These amounts assume that the DNA is in TE only ; if DNA is in a solution containing salt , adjust salt accordingly to achieve the correct final concentration . > 5 µL .
Mix well .
Add 2 to 2.5 volumes of cold 100 % ethanol ( calculated after salt addition ) . > 110 ( 2V ) .
Mix well .
Place on ice or at - 20 degrees C for > 20 minutes .
Spin a maximum speed in a microfuge 10 - 15 min .
Carefully decant supernatant .
Add 1 ml 70 % ethanol .
Mix .
Spin briefly .
Carefully decant supernatant .
Air dry or briefly vacuum dry pellet .
Resuspend pellet in the appropriate volume of TE or water .
