Silica-based Plasmid Miniprep (vacuum manifold)

Silica-based Plasmid Miniprep (vacuum manifold version)

Requirements

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

Method

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.

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

We 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.

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

References

J BROWN A rapid, non-toxic protocol for sequence-ready plasmid DNA Technical Tips Online (1997) 10.1016/S1366-2120(08)70079-5

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