Transgenic Mouse Facility

Preparation of transgene for microinjection: sucrose gradient method

The transgenic construct DNA should be free of vector sequences.

The plasmid preparation should be made from a Qiagen Endo free Kit or by the CsCl gradient centrifugation and then processed by restriction enzyme cuts to remove the plasmid vector sequences.

Finally, it should be purified as described below for microinjection.

The Facility has a stock of microinjection buffer available for final reconstitution of your transgene for microinjection.

Stock solutions

40% sucrose (100 ml)

  • 40 g sucrose
  • 5 ml 1 M Tris, pH 8.0
  • 20 ml 5 M NaCl
  • 2 ml 0.5 M EDTA

10% sucrose (100 ml)

  • 10 g sucrose
  • 5 ml 1 M Tris, pH 8.0
  • 20 ml 5 M NaCl
  • 2 ml 0.5 M EDTA

Sterilize both solutions using a separate 2.0 µm Nalgene filter with vacuum (this is important; you do not want your DNA to be chewed up!).

Major equipment

  • Gradient maker
  • Ultracentrifuge
  • SW41 swinging bucket rotor

Procedure

  1. Cut your transgene construct from your plasmid with the appropriate restriction enzymes.
    • Run an aliquot of the RE cuts on a checking gel to make sure the cut has gone to completion.
    • Note: Vector sequence and the transgene have to be clearly different sizes to separate efficiently on the gradient
  2. Using a gradient maker, prepare a linear gradient of 10-40% sucrose in ultra centrifuge tube.
    • Place equal volumes of sucrose in chambers with the 10% solution closed to the outlet.
      Place stirrer in 10% chamber.
    • Run tubing with 20 µL capillary pipette into ultracentrifuge tube.
    • Open gradient maker; layer gradient from bottom up (10% on bottom).
    • Carefully remove pipettes from ultracentrifuge tube — do not disturb gradient!
    • Place RE digested DNA on top of gradient (50-100 µL DNA).
    • Run SW41rotor at 30K RPM at 20°C for 16 hrs.
    • For calculating longer or shorter run times (T) or higher speed (RPM): T1(RPM1)2=T2(RPM2)2
  3. Place stopper with 2-syringe needle in top.
    • Carefully, puncture bottom of tube with needle and collect fractions (control speed with syringe or finger).
    • Collect 30-35 x 300 µL fraction s.
  4. Take a 15 µL aliquot from each fraction, run each aliquot in a separate well of a 1% agarose checking gel with suitable markers.
    • Document with picture.
    • Note: You can dilute the sample 1:2 to decrease the sucrose concentration.
  5. Pool the fraction of the correct size for the transgene.
  6. Dialyze the pooled fractions against sterile TE (10 mM Tris, pH 7.6, 0.25 mM EDTA)
  7. Concentrate transgene DNA.
    • Final preparation should be 8-10 ng/µL Transgene Microinjection buffer.

Reference

This protocol was obtained from Dr. C. Shashikant, Penn State University.