Erman's Lab:DNA Miniprep with Alkaline Lysis protocol
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Solutions/reagents:
- LB (+ antibiotics)
- <a name="Alkaline Lysis SLN1">Alkaline Lysis SLN1
<tab>(20mM Tris(pH 8), 50mM Glucose, 10mM EDTA)</a> - <a name="freshly prepared AL2">freshly prepared AL2
<tab>(0.2N NaOH, 1% SDS)</a> - <a name="AL3">AL3
<tab>(3M KAc, glacial acetic acid)</a> - isopropanol stored at room temperature
- 70% EtOH
- TE
- water
- single colony
Equipment:
- Incubator
- Centrifuge
- Eppendorf tubes
Steps:
- Inoculate 2 ml LB (+ antibiotics) with single colony and incubate with shaking for 12 hrs(overnight) at 37°C.
- Measure out 2 ml of culture into Eppendorf tube (1).
- Centrifuge at maximum speed for 1.5 mins at 4°C, gently aspirate out the supernatant and discard it.
- Leave pellet as dry as possible.
- Add 100 µl of <a href="#Alkaline Lysis SLN1" >Alkaline Lysis SLN1</a>.
Resuspend pellet by vortexing/by shaking vigorously. - Add 200 µl of <a href="#freshly prepared AL2" >freshly prepared AL2</a>.
Close the tube tightly and invert the tube 5 - 6 times.
Do not vortex! - Store the tube on ice.
- Add 300 µl of <a href="#AL3" >AL3</a>.
Close the tube tightly and gently mix the contents by inverting the tube. - Incubate on ice for 5 mins.
- Centrifuge at maximum speed for 5 mins at 4°C and aspirate out the top layer.
Transfer top aqueous layer into Eppendorf tube (2).
Discard bottom layer. - Measure out 900 µl of isopropanol into Eppendorf tube (2).
Vortex the mixture for a few secs. - Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
- Add 1 ml of 70% EtOH.
Vortex the mixture for a few secs. - Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
- Dry the pellet in air.
Option 1: Add 50 µl of TE.
(or)
Option 2: Add 50 µl of water.Dissolve the pellet in the solution.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 12 hrs, 26 mins
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