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Preparation of DNA for Microinjection

CWRU School of Medicine

Transgenic Mouse Core Facility

Preparation of DNA for Microinjection

  1. Restrict DNA and gel purify
    1. Cut 100ug DNA, removing as much plasmid sequence as possible from the insert.
    2. Run on an agarose gel.
    3. Cut out band and electroelute in 0.5X TBE.
    4. Add an equal volume of Tris saturated phenol with b-ME and hydroxyquinoline.
    5. Vortex, RTo X 3min.
    6. Add an equal volume of 24:1 CHCl3/Isoamyl Alcohol.
    7. Vortex, spin in clinical centrifuge at speed 5 for 5 min. at RTo. (between 1400-1600 G)
    8. Remove aq. layer to a new tube, discard organic layer.
    9. Repeat steps D-I two more times.
    10. Add an equal volume CHCl3, vortex, spin.
    11. Remove aq. to Millipore ultrafree MC filter units (0.22um)—you will need several. (#UFC30GV25 - pk. 25 - 0.5mL)
    12. Microfuge at speed 7, 1 min. at RTo.
    13. Remove the basket portion from the tubes.


  2. Ethanol precipitate
    1. Add 1/10th volume 3M NaOAc, pH=6.0 to aqueous layer.
    2. Add 2X volume EtOH.
    3. -20oC for at least 15 min.
    4. Microfuge at 4oC X 15 min.
    5. Remove EtOH.
    6. Air Dry.
    7. Resuspend each pellet in 50ul 10mM Tris, pH=7.5, 0.1mM EDTA. Combine into 1 tube.
    8. Re-precipitate by adding 1/10th volume 3M NaOAc, 2X volume EtOH.
    9. -20oC for at least 15 min.
    10. Microfuge at 4oC X 15 min.
    11. Remove EtOH.
    12. Wash with 0.5ml 70% EtOH.
    13. Microfuge at 4oC X 1 min.
    14. Remove EtOH, air dry pellet.


  3. Second filtering
    1. Resuspend pellet in 100ul 10mM Tris, 0.1mM EDTA.
    2. Put through a millipore basket filter as described above.


  4. Quantitate/Validate DNA
    1. Make a 1:50 dilution and read A260/A280 on spectrophotometer with the microcuvette. (Pure DNA A260/A280=1.8)
    2. Cut 1 ug insert DNA with a known enzyme and run on a gel alongside 1ug uncut insert DNA. Confirm that the DNA gives the expected pattern.
    3. Make a copy of the photo of this gel for the transgenic facility.
    4. Label the tube of DNA with the construct name , concentration, investigator, and date.
    5. Give tube to transgenic facility along with the photo of the gel.

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