Plasmid Preps

Silica-based Plasmid Miniprep (vacuum manifold version)

Reagents:

GTE:

50 mM Glucose, 25 mM Tris 8.0, 10 mM EDTA  RNAse A (0.1mg/ml)

Lysis Buffer:

200 mM NaOH, 1% SDS (prepare fresh)

4.0 M KOAc:

(pH 5.5 with acetic acid)

Silica Suspension:

resuspend immediately before use by shaking vigorously

Wash Solution:

50 mM NaCl, 10 mM Tris 7.5, 2.5 mM EDTA, 50% Ethanol

Procedure:

  1. Prepare Silica Suspension
    • suspend 2-10  grams of Silica (Sigma S-5631) in 20 mls H2O and allow to settle for 2 hours
    • remove milky supernatant and suspend settled silica in 20 mls H2O and allow to settle
    • repeat two more times
    • estimate volume and resuspend silica in 2 vols 6M Guanidine Hydrochloride-1M KOAc buffer, pH 5.5.
  2. Lyse bacteria
    • pellet 1.5 mls culture in microcentrifuge tube 10 sec @ 14,000 rpm
    • resuspend cells in 200 ul GTE
    • lyse cells with 200 ul lysis solution for 1 minute
    • add 200 ul KOAc and mix by inversion
    • remove debri by centrifugation 10 minutes @ 14,000 rpm
    • Put a cleaned Qiagen mini prep1 column on manifold (i.e. QIAvac 24 Cat#19403) and load  200ul of silica suspension. Do not have the vacuum on at this stage.
  3. Isolate plasmid DNA
    • Transfer the supernatant to the silica slurry and mix by pipetting.
    • Turn on vacuum and Wash 2X 1ml Wash solution.
    • Spin the column 1min high speed to dry (this is very important)
    • Elute the plasmid DNA with 50 to 100ul  H2O or EB.
      • yields approx 100-200 ng/ul plasmid DNA

  Notes, etc.

  • 1We use the used Qiagen columns from the mini prep and gel extraction kits. To clean these columns pass 1-2ml H20 (65C) followed by 0.5ml EtOH wash.
  • We generally use this procedure for mass mini preps (i.e. determining if you have cloned the correct insert). Once you have your clone you should prep some more using a new Qiagen column.

References:

  • A rapid, non-toxic protocol for sequence-ready plasmid DNA. J.D. Brown (1997). Technical Tips Online.
  • Extraction of superior-quality plasmid DNA by a combination of modified alkaline lysis and silica matrix. R. Lakshmi, et al. (1998). Analytical Biochemistry 272: 112-115.