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Method prepared by Grace Panganiban: gepangan@facstaff.wisc.edu

 

Injection DNA Preparation

1) Culture 400 mls of bacteria harboring transposon overnight at 37oC.

2) Harvest cells at 7000xg for 5 min. (pellet can be frozen at -80oC for several weeks)

3) Resuspend pellet thoroughly in 14 mls:

     for 32 mls       for 64 mls       for 128 mls       stock soln      50 mM glucose     1.6 mls         3.2 mls           1M      25 mM Tris 8.0    0.8 mls         1.6 mls           1M pH 8.0      10 mM EDTA        0.64 mls        1.28 mls          0.5 M 
4) Add 1.6 mls 10 mgs/ml lysozyme in above buffer.
Incubate 15-20 min at room temp.

5) Add:

     for 32 mls       for 64 mls       for 128 mls       stock soln      0.2 N NaOH       1.28 mls         2.56 mls          10 N      1% SDS           3.2 mls          6.4 mls           20% 
Swirl gently to mix. Incubate 5 min at room temp.

6) Add 16 mls cold 3M KOAc pH 4.8 (instructions for making this are in Appendix B of the Cold Spring Harbor cloning manual). Mix gently. Incubate 30 min. on ice. (Longer is okay)

7) Spin at 7000xg and 4oC for 10 min. Recover supernatant through a funnel lined with 2 layers of cheesecloth and into a 100 ml graduated cylinder. Add 0.6 volumes of isopropanol.

8) Centrifuge at 7000xg for 5-10 min. Drain. Air dry pellet (but not completely).

9) Resuspend pellet in 3 mls TE. Adjust volume to 4 mls with TE. Transfer to a 5 ml clear ultracentrifuge tube.

10) Add 3.8 g CsCl. Cover tube with Parafilm and shake until CsCl is dissolved. Add 0.4 mls of 10 mgs/ml EtBr. Balance tubes with TE or saturated CsCl soln.

11) Centrifuge at 40 K for 48 hrs in an SW-55 (or comparable) rotor.
Injection DNA Preparation (cont)

12) Pull lower (plasmid) band using a 22.5 gauge needle and a 1 ml syringe.

13) Extract EtBr with water saturated butanol or water saturated isopropanol.

13) Split sample into 2 (for safety) and dialyze vs. 4-5 changes of TE over ~24 hrs (dialysis buffer volume should be at least 100x sample volume).

14) Recover DNA from dialysis tubing, add 1/4 volume 10 M NH4Ac, and 2 1/2 volume absolute EtOH. Spin at 10,000xg for 10 min. Rinse pellet with 70% EtOH. Drain pellet and air dry (but not completely). Resuspend DNA in TE, determine concentration by A260-280 and store at 4oC.

15) Just prior to injection, mix 20 ug of transposon DNA with 20 ug pWC DNA, and EtOH ppt using 1/4 volume 1/4 volume 10 M NH4Ac, and 2 1/2 volume absolute EtOH. Rinse pellet with 70% EtOH. Drain pellet and air dry (but not completely). Resuspend DNA in 45 ul of injection buffer and 5 ul of (filtered) green food coloring.


Comments:
The CsCl banding protocol can be scaled up if necessary. I often pool 2-3 liters worth of DNA into 1 10 ml ultracentrifuge tube.

The DNA (especially for larger constructs) is much nicer if it is not dried completely after any of the alcohol ppt steps.