B. Midiprep double-stranded dna isolation

A midi-prep double-stranded DNA isolation has been developed to generate a sufficient amount of template DNA for several Sequenase[TM] catalyzed fluorescent terminator reactions. Here, one bacterial colony which harbored the plasmid of interest is picked into 3 ml of liquid media containing ampicillin and incubated in a 37degC shaker for 8-10 hours. At this time, the culture is transferred into 50 ml of ampicillin-containing media and incubated further for 10-12 hours. After harvesting the cells by centrifugation, a diatomaceous earth-based alkaline-lysis purification method (19-22) is performed, similar to that discussed above for large scale DNA isolation. The purified DNA is crudely assayed for concentration and purity by agarose gel electrophoresis against known standards. The approximate yield of double-stranded DNA using this method is 1 ug of DNA per ml of cell culture. For a 50 ml cell culture, about 50 ug of DNA are recovered, and 5 ug are used typically in a Sequenase[TM] terminator reaction.

Note: This procedure is the method of choice for isolating double stranded plasmid-based templates for the Sequenase Dye-Labeled Terminator Sequencing Reactions.

Protocol

1. Pick a colony of bacteria harboring the plasmid DNA of interest into a 12 X 75 mm Falcon tube containing 3 ml of 2xTY media supplemented with the appropriate antibiotic (typically ampicillin at 100 ug/ml) and incubate at 37deg C 8-10 hours with shaking at 250 rpm. Transfer the culture to an Ehrlenmeyer flask containing 50 ml of similar media, and incubate further for 11-14 hours.

2. Harvest the cells by centrifugation at 3000 rpm for 5 minutes in 50 ml conical tubes in the Beckman GPR tabletop centrifuge and decant the supernatant. The cell pellets can be frozen at -70degC at this point.

3. Resuspend the cell pellets in 2 ml of GET/Lysozyme solution, add 4 ml of alkaline lysis solution, gently mix, and incubate for 5 minutes in an ice-water bath.

4. Add 4 ml of 3M NaOAc, pH 4.8, gently mix by swirling, and incubate in an ice-water bath for 30-60 minutes.

5. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal DNA by pouring through a double-layer of cheesecloth into a new 50 ml conical tube. Centrifuge at 3,000 rpm for 20 minutes at 4degC in the Beckman GPR tabletop centrifuge.

6. Decant the supernatant to a 50 ml polypropylene centrifuge tube, add 20 ul of a 20 mg/ml DNase-free RNase A and incubate in a 37degC water bath for 30 minutes.

7. Add 7 ml (equal volume) of de-fined diatomaceous earth in guanidine-HCl (20 mg/ml) and allow the DNA to bind at room temperature for 5 minutes with occasional mixing. Centrifuge at 3,000 for 5 minutes in the Beckman GPR tabletop centrifuge.

8. Decant the supernatant, resuspend in 7 ml of diatomaceous earth-wash buffer, and centrifuge as above.

9. Decant the supernatant, resuspend in 7 ml of acetone, and centrifuge as above.

10. Decant the supernatant and dry in a vacuum oven.

11. Resuspend the pellet in 0.6 ml of 10:1 TE buffer, and elute the bound DNA by incubation at 65degC for 10 minutes with intermittent mixing.

12. Remove the diatomaceous earth by centrifugation at 3,000 rpm for 5 minutes in the in the Beckman GPR tabletop centrifuge.

13. Transfer the supernatant to a 1.5 ml microcentrifuge tube and centrifuge at 12,000 rpm for 5 minutes in a microcentrifuge at room temperature. Transfer the supernatant to a new 1.5 ml microcentrifuge tube and ethanol precipitate.

14. Resuspend the dried DNA pellet in 40 ul of 10:0.1 TE buffer and assay for concentration by agarose gel electrophoresis.