Cfrench:minipreps1: Difference between revisions
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==Plasmid DNA minipreps from ''Escherichia coli'' JM109 and similar strains | ==Plasmid DNA minipreps from ''Escherichia coli'' JM109 and similar strains== | ||
#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. | #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 1: 5 ul/ml of 5 mg/ml RNAse A (stock solution in freezer) in water: need 100 ul per prep. | ||
| Line 20: | Line 20: | ||
#Add 40 ul of EB (10 mM tris-HCl, pH8) and mix to redissolve the pellet. | #Add 40 ul of EB (10 mM tris-HCl, pH8) and mix to redissolve the pellet. | ||
#DNA should be stored at -20˚C. | #DNA should be stored at -20˚C. | ||
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Revision as of 02:55, 7 December 2006
Plasmid DNA minipreps from Escherichia coli JM109 and similar strains
- 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.
