Reagents :
STET (recipe?)
LB
5M NH4Ac
absolute alcohol
70% alcohol
Lysozyme (conc?)
Procedure :
1, Prepare a 2 ml overnight baterial culture (in a STERILIN TUBE).
2, Spin down the cell, discarded the supernant and resuspend the cell in 200ml STET.
3, To an eppendorf tube, add 20ml lysozyme*.
4, With the help of a 1ml pipeman, transfer the cell to the eppendorf tube.
5, Mix the content gently and make a pin hole on the cover.
6, Boil in a boiling bath for 60-90 sec..
7, Spin (max. speed) for 5min.
8, Reove the pellet carefully with a toothpick.
9, Add 150ml NH4Ac and 1ml absolute alcohol to the supernatant.
10, Mix well and store in -20oC for at least 20min..
11, Spin for min 20 min. (max. speed)
12, Discard supernant and wash the pellet with 70% alcohol.
13, Air dry, or dry in a speed vac for 10-15min if necessary.
14, If the wall of the tube is too wet, clean it with a cotton buds.
15, Redissolve the pellet in 20-25ml water.
16, Use 1ml for Enzyme Digestion screening.
* Lysozyme has to be prepared freshly on day of use.
Conc = 10 mg/ml
Alternatly, 8mg/ml can be used. Use 25ml for each sample.
Usually 1-2 mg of DNA can be prepared by this method.
Yield can be increased by starting with a larger volume of baterial culture (Max.=5ml).