Materials
- GTE buffer (50mM glucose, 25mM Tris-HCl (pH 8.0), 10mM EDTA (pH 8.0), (200μl tube)
- 0.2 N NaOH / 1% SDS (freshly made) (300μl/tube)
- 3.0M Potassium acetate (pH 4.8) (300μl/tube)
- RNase A (DNase-free)(10mg/ml) (2μl/tube)
- Chloroform (400μl/tube)
- Isopropanol, 100%
- Ethanol, 70%
- 4.0 M NaCl
- 13% PEG8000 (sterilized by autoclaving, rather than by filtration)
- Deionzed H2O
- Terrific Broth (1L)
To prepare Terrific Broth, add 100ml of a sterile solution of 0.17 M KH2PO4 and 0.72 M K2HPO4 to 900ml of base broth (base broth = 12g bacto-tryptone, 24g bacto-yeast extract, 4.0 ml glycerol, q.s. to 900ml with Deionzed H2O and then autoclave).
* Note : The above volume of glycerol is most easily measured out by weighing: 4.0ml glycerol = 5.0g.
Methods
- Incubate cultures overnight at 37°C in Terrific Broth, with an appropriate amount of antibiotic, in Erlenmeyer flasks or 50ml polypropylene tubes. (To maintain adequate aeration in the flasks or tubes, restrict the culture volume to be no more than one quarter of the total flask volume, or one fifth of the total tube volume.)
- IPellet 1.5ml aliquots of culture for 1min in a microcentrifuge. *Note : A total culture volume of 4.5 ml can be spin down per tube without change volumes in the procedure. This allows you to achieve a three-fold increase in yield while eliminating the need for extra tubes and additional handling.
- Remove the supernatant by aspiration and re-suspend the bacterial pellet in 200μl of GTE buffer by pipetting up and down.
- Add 300μl of freshly prepared 0.2 N NaOH / 1% SDS and then mix the contents of the tube by inversion until the solution clears. Then incubate on ice for 5 min.
- INeutralize the solution by adding 3003.0 M potassium acetate, pH 4.8, mix μl of by inverting the tube, and incubate on ice for 5 min.
- Remove cellular debris by centrifuging for 10 min at room temperature, and then transfer the supernatant to a clean tube.
- Add RNase A (DNase-free) to a final concentration of 20 g/ml and incubate the tube at 37°C for 20 min.
- After the RNase A treatment, extract the supernatant twice with 400μl of chloroform. Mix the layers by hand for 30 s after each extraction. Centrifuge the tube for 1 min to separate the phases and remove the aqueous phase to a clean tube. *Note : Inadequate extraction will result in poor sequencing data. An additiona CHCl3 extraction may be necessary if debris is still visible at the interface after the second extraction.
- Precipitate the total DNA by adding an equal volume of 100% isopropanol and immediately centrifuging the tube for 10 min at room temperature.
- Wash the DNA pellet with 500μL of 70% ethanol and then dry under vacuum for 3 min.
- Dissolve the pellet in 32μl of deionized H2O and precipitate the plasmid DNA by first adding 8.0μl of 4M NaCl, and then adding 40μL of autoclaved 13 % PEG8000
- After thorough mixing, incubate the sample on ice for 20 min, and then pellet the plasmid DNA by centrifugation for 15 min at 4°C in a fixed-angle rotor. *Note1:The temperature parameter here is very important; adhere to the recommended 4°C. *Note 2 : If you use a horizontal rotor, do not aspirate the supernatant in Step 13 because the clear pellet adheres to the bottom of the tube and can be lost if you are not careful. Remove the supernatant by decanting. Eitherapproach, decanting or aspirating, can be used to remove the supernatant from a tube spun in a non-horizontal rotor.
- Carefully remove the supernatant and rinse the pellet with 500l of 70% ethanol. Then dry the pellet under vacuum for 3 min, resuspend in 20μl of deionized H2O, and store at -20°C.
