Preparation of plasmid DNA （质粒制备）

Preparation of plasmid DNA : a modified mini alkaline-lysis/PEG precipitation procedure. ABI; 1995

Materials

GTE buffer (50mM glucose, 25mM Tris-HCl (pH8.0), 10mM EDTA (pH 8.0)) (200µl/tube)

0.2N NaOH / 1% SDS (freshly made) (300µl/tube)

3.0M Potassium acetate (pH4.8) (300µl/tube)

RNase A (DNase-free) (10 mg/ml) (2µl/tube)

Chloroform (400 µl/tube)

Isopropanol, 100%

Ethanol, 70%

4.0 M NaCl

To prepare Terrific Broth, add 100 ml of a sterile solution of 0.17M KH2PO4 AND 0.72M K2HPO4 TO 900 ml of base broth (base broth = 12g bacto-tryptone, 24g bacto-yeast extract, 4.0 ml glycerol, q.s. to 900 ml with deionized water and then autoclave).

Note: The above volume of glycerol is most easily measured out by weighing : 4.0ml glycerol = 5.0 g.

Methods

1.Incubate cultures overnight at 37ºC 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.)

2.Pellet 1.5 ml aliquots of culture for 1 minute in a microcentrifuge.

Note: A total culture volume of 4.5 ml can be spun down per tube without changing volumes in the procedure. This allows you to achieve a three-fold increase in yield while eliminating the nedd for extra tubes and additional handling.

1.Remove the supernatant by aspiration and resuspend the bacterial pellet in 200 µl of GTE buffer by pipetting up and down.

2.Add 300µl of freshly prepared 0.2N NaOH / 1% SDS and then mix the contents of the tube by inversion until the solution clears. Then incubate on ice for 5 minutes.

Note: Throughout this procedure, the use of a vortex must be avoided so as to minimize shearing of the contamination chromosomal DNA .

1.Neutralize the solution by adding 300µl of 3.0 M potassium acetate, pH 4.8, mix by inverting the tube, and incubate on ice for 5 minutes.

2.Remove cellular debris by centrifuging for 10 minutes at room temperature, and then transfer the supernatant to a clean tube.

3.Add RNase A (DNase-free) to a final concentration of 20µg/ml and incubate the tube at 37ºC for 20 minutes.

4.After the RNase A treatment, extract the supernatant twice with 400µl of chloroform. Mix the layers by hand for 30 seconds after each extraction. Centrifuge the tube for 1 minute to separate the phases and remove the aqueous phase to clean tube.

Note: Inadequate extraction will result in poor sequencing data. And additional chloroform extraction may be necessary if debris is still visible at the interface after the second extration.

1.Precipitate the total DNA by adding and equal volume of 100% isopropanol and immediately centrifugeing the tube for 10 minutes at room temperature.

2.Wash the DNA pellet with 500µl of 70% ethanol and then dry under vacuum for 3 minutes.

3.Dissolve the pellet in 32µl of deionized water and precipitate the plasmid DNA by first adding 8.0 µl of 4M NaCl, and then adding 40µl of autoclaved 13% PEG8000.

4.After thorough mixing, incubate the sample on ice for 20 minutes, and then pellet the plasmid DNA by centrifugation for 15 minutes at 4ºC in a fixed angle rotor.

Note 1: The temperature parameter here is very important; adhere to the recommended 4ºC.

Note 2: If you use a horizontal rotor, do not aspirate the supernatant in the next step 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. Either approach, decanting or aspiration can be used to remove the supernatant for a tube spun in a non-horizontal rotor.

1.Carefully remove the supernatant and reinse the pellet with 500µl of 70% ethanol. Then dry the pellet under vacuum for 3 minutes, resuspend in 20µl of deionized water and store at -20º C.