Maxiprep of plasmid DNA from E.coli protocol
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Solutions/reagents:
- LB broth + selective marker
- 50% sterile glycerol
- <a name="TEG">TEG
<tab>(25mM Tris-Cl, 10mM EDTA, 50mM dextrose)</a> - 20 mg/ml lysozyme
- 10% SDS
- 4M NaOH
- autoclaved water
- <a name="Solution 3">Solution 3
<tab>(3M potassium-acetate, 2M acetic acid -- glacial is 17M)</a> - isopropanol
- 70% ethanol
- TE buffer
- 5M LiCl
- 1 mg/ml RNaseA
- <a name="phenol: chloroform: isoamyl alcohol">phenol: chloroform: isoamyl alcohol
<tab>(25:24:1)</a> - <a name="chloroform: isoamyl alcohol">chloroform: isoamyl alcohol
<tab>(24:1)</a> - straight ethanol
- 3M sodium acetate
- a single colony of E. coli
Equipment:
- Incubator
- Centrifuge
- Flasks of appropriate volumes
- Eppendorf tubes
- Oakridge tubes
Steps:
- Inoculate LB broth + selective marker with a single colony of E. coli and incubate with shaking for 12 hrs(overnight) at 37°C.
- Set aside a fresh an Eppendorf tube. Call it Eppendorf 1.
Measure out 850 µl of culture into Eppendorf 1.
Add 150 µl of 50% sterile glycerol.
Store at -80°C.
Set aside a fresh an Eppendorf tube. Call it Eppendorf 2.
Measure out 850 µl of culture into Eppendorf 2.
Add 150 µl of 50% sterile glycerol.
Store at -80°C. - Measure out as much culture as will fit into an Oakridge tube.
Centrifuge at a speed of 5800 Xg for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
Add rest of the culture to pellet.
Centrifuge at a speed of 5800 Xg for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
Add 1 ml of <a href="#TEG" >TEG</a>.
Resuspend the pellet by vortexing/by shaking vigorously. - Add 111 µl of 20 mg/ml lysozyme.
Incubate on ice for 30 mins. - Meanwhile:
Measure out 250 µl of 10% SDS into an Eppendorf tube.
Add 125 µl of 4M NaOH.
Add 2.125 µl of autoclaved water.
Vortex the mixture for a few secs. - Measure out 2 ml of SDS/NaOH mix into the Oakridge tube.
Incubate on ice for 10 mins. - Add 1.5 ml of <a href="#Solution 3" >Solution 3</a>.
Incubate on ice for 10 mins. - Vortex the mixture for a few secs.
Centrifuge at a speed of 17200 Xg for 15 mins at 4°C and aspirate out the top layer.
Transfer top aqueous layer into an Oakridge tube.
Discard bottom layer. - Add 2.7 ml of isopropanol.
Centrifuge at a speed of 17200 Xg for 10 mins at room temperature, gently aspirate out the supernatant and discard it. - Add 1 ml of 70% ethanol.
Vortex the mixture for a few secs.
Centrifuge at a speed of 17200 Xg for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
Dry the pellet in air for 2 - 5 mins.
Add 500 µl of TE buffer.
Resuspend the pellet by vortexing/by shaking vigorously.
Add 500 µl of 5M LiCl.
Incubate on ice for 5 mins.
Centrifuge at a speed of 17200 Xg for 10 mins at room temperature and aspirate out the top layer.
Transfer top aqueous layer into an Eppendorf tube.
Discard bottom layer. - Add 1 ml of isopropanol.
Incubate at room temperature for 10 mins. - Centrifuge at a speed of 17200 Xg for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
- Add 100 µl of 70% ethanol.
Vortex the mixture for a few secs.
Centrifuge at a speed of 17200 Xg for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
Add 375 µl of TE buffer.
Resuspend the pellet by vortexing/by shaking vigorously.
Add 7.5 µl of 1 mg/ml RNaseA.
Incubate at 37°C for 30 mins. - Add 700 µl of <a href="#phenol: chloroform: isoamyl alcohol" >phenol: chloroform: isoamyl alcohol</a>.
Vortex the mixture for a few secs.
The solution should be thoroughly mixed.
Centrifuge at maximum speed for 2 mins at room temperature and aspirate out the top layer.
Transfer top aqueous layer into an Eppendorf tube.
Discard bottom layer. - Repeat protocol from Step 14 until the interface between the phases is clear after centrifugation.
- Add 700 µl of <a href="#chloroform: isoamyl alcohol" >chloroform: isoamyl alcohol</a>.
Vortex the mixture for a few secs.
The solution should be thoroughly mixed.
Centrifuge at maximum speed for 2 mins at room temperature and aspirate out the top layer.
Transfer top aqueous layer into an Eppendorf tube.
Discard bottom layer. - Repeat Step 16.
This removes phenol. - Add 750 µl of straight ethanol.
Add 125 µl of 3M sodium acetate.Option 1: Store at -80°C for 30 mins.
(or)
Option 2: Store at -20°C for 12 hrs(overnight). - Centrifuge at a speed of 13600 Xg for 15 mins at 4°C, gently aspirate out the supernatant and discard it.
Add ~100 µl of 70% ethanol.
Vortex the mixture for a few secs.
Centrifuge at a speed of 13600 Xg for 5 mins at 4°C, gently aspirate out the supernatant and discard it.
Add 100 - 200 µl of TE buffer.
Resuspend the pellet by vortexing/by shaking vigorously.
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