Difference between revisions of "Cfrench:minipreps1"
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#DNA should be stored at -20˚C.
#DNA should be stored at -20˚C.
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Latest revision as of 08:10, 8 July 2007
Plasmid DNA minipreps from Escherichia coli JM109 and similar strains
0.4 M NaOH. Dissolve 1.6 g solid NaOH in 100 ml water. Note: NaOH is caustic! Wear gloves and eye protection! Store at room temperature.
10% w/v SDS. Dissolve 5 g solid SDS (sodium dodecyl sulphate, also known as sodium lauryl sulphate) in 47 ml water. SDS is an irritant. Wear gloves and eye protection. Store at room temperature. On cold days, SDS may precipitate; warm the bottle gently or microwave it briefly on low to medium power to redissolve it.
5 mg/ml RNAse A in water. Dissolve 5 mg lyophilised RNAse A powder (Sigma) in 1 ml water. Store at -20˚C.
Solution 3: 3 M potassium acetate + 2 M acetic acid. Dissolve 29.45 g solid potassium acetate (fw 98.15) in about 80 ml water, and add 11.45 ml glacial acetic acid (fw 60.05, d 1.049). Acetic acid is corrosive and volatile, with a strong odour. Wear gloves and eye protection and work in a fume hood when handling glacial acetic acid. I suggest using a P5000 pipette to transfer 3 x 3.82 ml acetic acid. Rinse the pipette tip prior to disposal. Make up the final volume of the solution to 100 ml with water. Store at 4˚C (this solution is not temperature labile, but needs to be cold when it is used).
EB (elution buffer): 10 mM Tris buffer, pH 8. To make 100 ml of EB, dissolve 53 mg Tris base and 88 mg Tris hydrochloride in 100 ml water. Sterilise by autoclaving.
- Grow 3 ml overnight culture of E. coli with appropriate antibiotics. Note: if using the pre-prepared 5 ml bottles from the media suite, remove 2 ml of medium before inoculating; otherwise the culture will become anoxic and the plasmid DNA will be of poor quality.
- Prepare solution 1: 5 ul/ml of 5 mg/ml RNAse A (stock solution in freezer) in water: need 100 ul per prep.
- Prepare solution 2:
- for 4 preps: 0.4 ml water + 0.5 ml 0.4 M NaOH + 0.1 ml 10% SDS
- for 6 preps: 0.56 ml water + 0.7 ml 0.4 M NaOH + 0.14 ml 10% SDS
- Safety Note: 0.4M NaOH is caustic. Wear gloves and eye protection
- Transfer 1.3 ml culture to microcentrifuge tubes and harvest at medium speed (8 000 rpm) for 3 min.
- Remove supernatant (to autoclave waste), add 0.1 ml Solution 1 to pellet, and resuspend cells by pipetting up and down several times.
- Add 0.2 ml solution 2. Mix by inversion several times. The suspension should become clear and viscous.
- Add 0.15 ml solution 3 (in refrigerator) and mix immediately by inversion. A prepipitate should form. Solution 3 is 3M potassium acetate plus 2M acetic acid in water, and should be chilled before use.
- Incubate on ice for 10 min, mixing again at 5 min.
- Spin at high speed (12 000 rpm) for 3 min.
- Transfer supernatant (should be around 0.42 ml) to fresh tube and discard pellet to autoclave waste. The pellet is likely to be loose and have floating bits; as far as possible, try to avoid transferring any of these to the fresh tube.
- Add 0.92 ml ethanol and mix by inversion (if you recovered significantly less than 0.42 ml of supernatant, use about 2.2 volumes of ethanol).
- Incubate on ice for 10 min or in freezer overnight.
- Spin at high speed for 10 min. The DNA should form a pellet which may or may not be visible.
- Remove and discard the supernatant. Be careful not to disturb the pellet, or, if the pellet is invisible, be careful not to disturb the place where it ought to be.
- Add 0.2 ml of 70% ethanol, respin briefly, and remove and discard supernatant, being careful not to disturb the pellet. If necessary, respin to remove any liquid that may be clinging to the walls of the tube.
- Add 40 ul of EB (10 mM tris-HCl, pH8) and mix to redissolve the pellet.
- DNA should be stored at -20˚C.