Mini-preps

 

Stuff you need:

TENS Buffer

• 10 mM Tris-HCl pH 8.0
  1 mM EDTA pH 8.0
  0.1 N NaOH
  0.5% SDS
  

TE pH 8.0

• 10 mM Tris-HCl pH 8.0
  1 mM EDTA pH 8.0
  

95% EtOH precooled to -20 deg C

70% EtOH at room temperature

3 M Na Acetate pH 5.2

1. Spin 1.5 ml of overnight culture for 10 s in a microcentrifuge to pellet cells.
2. Decant supernatant, leaving 50-100 µl of media with pellet. Vortex thoroughly to resuspend cells.
3. Add 300 µl TENS, vortex on high 2-5 s.
4. Add 150 µl 3 M Na Acetate pH 5.2, vortex on high 2-5 s.
5. Spin 4 min to pellet cell debris and chromosomal DNA.
6. Transfer supernatant (~450 µl) to a fresh tube (you can usually do this by simply pouring the supernatant) and mix well with 0.9 ml 95% EtOH which has been precooled to -20 deg C.
7. NOTE: If the strain you are working with is not endA- (e.g. MC1066, JF1754 or TG1/lambda), it is recommended to do a 1:1 phenol:chloroform extraction before adding the ethanol.
8. Spin 2 min at room temperature to pellet plasmid DNA.
9. Discard supernatant, and wash twice with 70% EtOH (room temperature). Washing simply involves gently adding some 70% EtOH (using a squirt bottle, for example), and then pouring it off.
10. Resuspend pellet in 200 µl TE pH 8.0
11. Use 10 µl per digest and be sure to use RNase during digestion.