Plasmid mini-prep with diatomaceous earth

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1. preparation of buffers

• 100 ml resuspension buffer B1
  • 9 g glucose
  • 4 ml 0.25 M EDTA pH 8.0
  • 2.5 ml 1 M TRIS/Cl pH 8.0
  • bi-destillated water (ABD)
• 10 ml fresh lysis buffer B2
  • 500 µl 20% SDS
  • 200 µl 10 M NaOH
  • ABD
• neutralization buffer B3
  • 3 M sodium acetate pH 5.2
• DNA binding buffer B4
  • shake up 5 mg of diatomaceous earth (Sigma D5384) in 100 ml ABD and let it settle for 3 hours
  • remove the supernatant containing fine particles
  • repeat this step
  • add ABD to 100 ml (and EDTA to 20 mM if you want) and store the suspension at 4°C
  • prepare 6 M guanidine hydrochloride (GnHCl) in ABD
  • add 2 ml of the resuspended diatomaceous earth to 50 ml 6M GnHCl
• 80% isopropanol
• aceton
• 10 mM TRIS/Cl pH 7.5 - 8.5

2. plasmid prep

• grow bacteria in 2 ml 2xTY over-night
• centrifuge 30 sec
• remove supernatant
• completely resuspend bacteria in 200 µl B1
• add 200 µl lysis buffer B2 and mix gently
• add 300 µl neutralization buffer B3
• centrifuge 5 min
• transfer the supernatant to new tubes and mix with 500 µl DNA binding buffer B4 by vortexing
• centrifuge 5 min
• remove supernatant and wash diatomaceous earth pellet with 1 ml 80% isopropanol
• centrifuge 5 min
• remove isopropanol and wash pellet with 1 ml aceton
• centrifuge 5 min
• remove acetone and let pellet dry. Do not overdry.
• add 60 µl 10 mM TRIS/Cl pH 7.5 - 8.5 and vortex
• centrifuge 5 min
• transfer 50 µl of the supernatant to a new tube
• use 10 µl for analytical digest

3. principle

DNA binds in presence of chaotropic salts (guanidine hydrochloride, guanidin isocyanate, etc.) and at a pH of <7 to silicates. ("...glass binds DNA." (Vogelstein & Gillespie, P.N.A.S. 76 (1979): 615-619)) To elute the DNA it is important to remove the salt and to rise the pH to >7.5.