Protocols

Preparation of BAC DNA for Pronuclear Injection

(Courtesy of Dr. Douglas Epstein, Department of Genetics, University of Pennsylvania School of Medicine)

Reagents
Resuspension buffer: 50 mM Tris-Cl, pH 8.0, 10 mM EDTA
Lysis buffer: 200 mM NaOH, 1% SDS
Neuralization buffer: 3.0 M potassium acetate, pH 5.5
TE buffer: 10 mM Tris-Cl, pH 8.0, 1 mM EDTA
Microinjection buffer: 10 mM Tris-Cl, pH7.4, 0.1 mM EDTA
DNA Quanti-Ladder (Origene)
PI-SceI (New England Biolabs)

Procedure

There are many protocols available to harvest BAC DNA. We have tried several of them and prefer the old fashioned miniprep.

  1. Inoculate a single bacterial colony containing the BAC of interest into 10~20 ml of LB medium with the appropriate antibiotic(s) and incubate with shaking (225 rpm) at 37°C overnight.
  2. Split culture into 5~10 microcentrifuge tubes, harvest the cells by centrifugation 1 min at 10,000 x g and resuspend the pellet in 200 ul of the resuspension buffer containing fresh RNase A (100 ug/ul).
  3. Add 200 ul of the lysis buffer, and mix by gently inverting. No incubation time is necessary. Vortexing or vigorous shaking at this point will cause E.coli chromosomal DNA contamination.
  4. Add 200 ul of the neutralization buffer and mix by gently inverting. No incubation time is necessary.
  5. Centrifuge the mixture 10 min at 12000 x g, at 4°C.
  6. Transfer the supernatant to a new microcentrifuge tube and add 500 ul of Phenol/Chloroform/Isoamyl alcohol, pH6.7 (25:24:1). Vortex 5 sec (supercoiled BAC DNA does not shear) and centrifuge 2 min at 12000 x g at room temperature.
  7. Transfer the upper phase to a new tube and add 500 ul of chloroform. Vortex 5 sec and centrifuge 2 min at 12000 x g at room temperature.
  8. Transfer the upper phase to a new tube and add 900 ul of ethanol.
  9. Centrifuge the mixture for 30 min at 12000 x g at 4°C
  10. Wash the pellet with 75% ethanol, remove the ethanol and let the pellet air dry for 5-10 min. Resuspend the pellet in 20 ul of TE buffer.
  11. Pool DNA samples into a single microcentrifuge tube.
  12. Adjust the volume to 400 ul with TE buffer, add 40 ul of sodium acetate (3M, pH7.0) and 800 ul of ethanol to the tube, and mix by gentle inverting. The DNA will immediately precipitate and form an aggregate. This step will remove most, but not all, of the contaminating bacterial RNA.
  13. Immediately transfer the aggregate to a new tube using a pipette tip with a large orifice, and pulse-spin it down to the bottom of the tube.
  14. Dissolve the pellet in 500 ul TE, add 1 ug of fresh RNase A to the tube and incubate for 30 min to 1h at room temperature. This step is necessary to remove the remaining trace amounts of bacterial RNA.
  15. Add 500 ul of Phenol/Chloroform/Isoamyl alcohol, pH6.7 (25:24:1). Vortex 5 sec and centrifuge 2 min at 12000 x g at room temperature.
  16. Transfer the upper phase to a new tube and add 500 ul of Chloroform. Vortex 5 sec and centrifuge 2 min at 12000 x g at room temperature.
  17. Transfer the upper phase to a new tube and add 50 ul of sodium acetate (3M, pH7.0) and 900 ul of ethanol.
  18. Centrifuge the mixture for 30 min at 12000 x g at 4°C. Wash the pellet with 75% ethanol, let it air dry and resuspend in 10 ~ 20 ul of the microinjection buffer. Store the BAC DNA at 4°C. Do not freeze because repeated freezing and thawing will cause shearing.
  19. Estimate the BAC DNA concentration by running an aliquot on a 0.8 % agarose gel with a known amount of size markers. Immediately prior to microinjection, dilute an aliquot of DNA to a final concentration of 0.5 ~ 1 ng/ul using the microinjection buffer.
  20. (optional) Although circular BAC DNA can be used for microinjection we prefer to control the linearization site by digesting the BAC DNA with PI-SceI. The recognition site for PI-SceI is extremely rare occurring once in every 7 x 1010 base pairs of random sequence. It has been incorporated into the backbone of BAC vectors derived from CHORI.
    • Mix 2ug of BAC DNA, 2 ul of 10 x Reaction buffer, 2 units of PI-SceI (NEB) and water to a final volume of 20 ul. Incubate at least 3 h at 37°C and heat-inactivate for 20 min at 65°C.
    • Add 80 ul of water, 15 ul of sodium acetate (3M, pH7.0) and 300 ul of ethanol to the reaction mixture and centrifuge 30 min at 12000 x g at 4°C.
    • Wash the pellet with 75% ethanol, let it air dry, and resuspend the DNA in 20 ul of the microinjection buffer. The concentration should be ~100 ng/ul, and immediately before injection, dilute an aliquot to a final concentration of 0.5 ~ 1 ng/ul using the microinjection buffer.