Plasmid DNA Isolation
DNA Isolation from 2 ml cultures
This is a rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a standard method that uses no organic extractions or columns. The method works very well for doing analytical restriction digests of PAC clones and can be scaled up if necessary. With slight alterations this procedure can also be used for routine analysis of M13 RF, plasmid and cosmid DNAs.
I. Solutions
P1 (filter sterilized, stored at 4 C)
50 mM Tris, pH 8.0 (or 25 mM Tris-HCl, pH8)
10 mM EDTA, pH8.0
100 mg /ml RNase A
(1 M Tris-HCl 25 ml, 0.5 M EDTA 10 ml, ddH2O to 500 ml)
P2 (filter sterilized, stored at room temp)
0.2N NaOH
1% SDS
( 2N NaOH 1 ml, 10% SDS 1 ml, ddH2O to 10 ml)
P3 (autoclaved, stored at room temp)
3M KOAc, pH 4.8-5.5
(5M KAc 300 ml, Glacial acetic acid 57.5 ml, ddH2O to 500 ml)
II. Method
1. Using a sterile toothpick, inoculate a single isolated bacterial colony into 2 ml LB media supplemented with 25 mg/ml kanamycin (use 12.5 mg/ml chloramphenicol if using pCYPAC4 or BAC clones). Use a 12-15 ml snap-cap polypropylene tube. Grow overnight (up to 16 h) shaking at 225-300 rpm at 37 C.
2. Transfer 1.5 ml of culture to a 2 ml eppendorf tube and centrifuge for 30 seconds.
3. Discard supernatants. Resuspend (vortex) each pellet in 0.3 ml P1 solution. Add 0.3 ml of P2 solution and gently shake (invert) tube to mix the contents. The appearance of the suspension should change from very turbid to almost translucent.
4. Slowly add 0.3 ml P3 solution to each tube. A thick white precipitate of protein and E. coli chromosomal DNA will form. Invert the tube several times to mix the solution. Place tube on ice, 5 min (or longer).
5. Spin in a microcentrifuge for 10 min, to pellet the white precipitate.
6. Remove tubes from centrifuge; transfer ~.7-.8 ml of supernatant using a P1000 to a 2 ml eppendorf tube. (Note -- If it appears that some white precipitate was transferred over, re-spin for 5 min and transfer to a new tube.) Add 0.8 ml ice-cold isopropanol. Mix by inverting tube several times. At this stage, samples can be left at -20 C overnight.
7. Spin in microfuge (room temp or 4 C) for 15 min.
8. Remove supernatant and add 0.5 ml of 70% EtOH to each tube. Invert tubes several times to wash the DNA pellets. Spin in microfuge for 5 min. Optional -- repeat step 8.
9. Remove as much of the supernatant as possible. Occasionally, pellets will become dislodged from tube so it is better to carefully aspirate off the supernatant rather than pour it off.
10. Air dry pellets at room temp (do not SpeedVac as this results in over-drying and makes resuspension of sample difficult). When the DNA pellets turn from white to translucent in appearance, i.e., when most of the ethanol has evaporated, resuspend each in 40 ml TE. Allow the solution to sit in the tube with occasional tapping of the bottom of the tube (or by gentle vortexing).
11. Use 3-5 ml for digestion with NotI or other rare cutter enzymes. There are NotI sites flanking the Sp6 and T7 promotor regions of the pCYPAC-2 and derivative vectors; therefore, this is a very useful enzyme for analysis of insert size and for partial digest restriction mapping. Use 7-10 ml for digestion with a more frequent cutter such as BamHI or EcoRI.
Isolation of PAC DNA from 100 ml cultures
In contrast to multi-copy replicon systems, DNA preparations from PAC (BAC) cultures often contain comparatively high quantities of E. coli chromosomal DNA. While Qiagen- (or other column-) purified PAC preparations are relatively devoid of E. coli DNA, the yields are often very low. The method described here does not use any columns and is essentially a scaled-up version of the 2 ml miniprep method (above). In order to remove any contaminating E. coli chromosomal DNA, we employ the "Plasmid-Safe" ATP-dependent DNase (RecBC) enzyme from Epicentre Technologies, which digests away all DNAs except those that are circularized. The resultant DNA is sufficient for subcloning and restriction analysis. This method can be scaled up if necessary.
1. Inoculate 100 ml of LB-kanamycin (25 mg/ml) with a 1 ml pre-inoculum of the desired PAC clone. Use a 500 ml erlenmeyer flask and grow at 37 C in an environmental shaker at 300 rpm.
2. Spin down using GSA bottle, 5000 rpm, 5 min, ~ 4 C. Completely discard supernatant.
3. Suspend with 8 ml of P1 solution (use a serological pipet to resuspend the chunks of material). Transfer to a 50 ml SA600 (SS34) screw-top polypropylene tube. Vortex briefly to completely suspend the bacteria.
4. Add 8 ml of P2 solution. Gently swirl to mix. Allow to sit on lab bench for five minutes, swirling occasionally.
5. Add 8 ml of P3 solution. Invert a few times to mix. Place tube on ice for 10 min.
6. Spin in SA600 (SS34) rotor for 15 min at 12,000 rpm, 4 C.
7. Carefully transfer the supernatant to another SA600 (SS34) screw-top tube. Add an equal volume of ice-cold isopropanol and mix gently. Place on ice for 10 min.
8. Spin using SA600 (SS34) rotor for 15 min at 12,000 rpm, 4 C.
9. Discard supernatant (use a P200 to remove all traces of the isopropanol). Add 5 ml of 70% ETOH and invert the tube several times to wash the pellet.
10. Spin using SA600 (SS34) rotor for 5 min at 12,000 rpm, 4 C.
11. Discard supernatant completely (use a P200 to remove all traces of the 70% ETOH). Air-dry briefly until the pellet turns translucent. Add 500 ml 1x plasmid safe buffer (see below) and swirl several times to resuspend the DNA. Add 5 ml of 100 mM ATP, 5 ml of plasmid-safe ATP-dependent DNase (aka RecBC; Epicentre Technologies), 1 ml of RNase (10 mg/ml). Place the tube in a 37 C water bath for 3 hours with occasional swirling.
12. Transfer the DNA to a 2 ml eppendorf tube.
13. Phenol-extract the sample, 1 min, gentle vortexing (or tube inversion). Spin in microfuge, 1 min. Discard bottom (organic) layer.
14. PCI-extract the sample, 1 min, gentle vortexing (or tube inversion). Spin in microfuge, 1 min. Discard organic layer. Repeat step 14 if necessary (i.e., if lots of white interface remains).
15. CI-extract the sample, 1 min, gentle vortexing (or tube inversion). Spin in microfuge, 1 min.
16. Add 50 ml of 3 M NaOAc and 1.1 ml of absolute ETOH. Place on ice, 10 min. Spin in microfuge, 10 min.
17. Completely remove the supernatant. Add 1.8 ml 70% ETOH and invert tube several times. Spin in microfuge, 1 min.
18. Remove the supernanant (use a P20 to completely remove all traces of ETOH). Air-dry briefly.
19. Add 400 ml of TE and tap the bottom of the tube a few times. Allow the tube to sit at room temp. Let the DNA go into solution on its own (do not vortex).
10X Plasmid-safe ATP-dependent DNase reaction buffer: 330 mM Tris-Acetate (pH ~8), 660 mM potassium acetate, 100 mM magnesium acetate, 5 mM DTT. Add all components except DTT. Autoclave, cool down, add DTT. Store at 4 C.
