Miniprep - GET Buffer protocol
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Solutions/reagents:
- overnight culture
- <a name="ice-cold GET buffer"> ice-cold GET buffer
<tab>(50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8)</a> - 0.2M NaOH stored at room temperature
- <a name="ice-cold potassium acetate solution"> ice-cold potassium acetate solution
<tab>(3 M potassium acetate, 1.8 M acetic acid, no pH adjustment)</a> - 95% / 100% ethanol
- 70% EtOH
- TE buffer
- distilled water
- <a name="PCA solution">PCA solution
<tab>((optional)50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol)</a> - SDS
- <a name="lysozyme">lysozyme
<tab>(optional)
</a>
Equipment:
- Centrifuge
- Sterile 2-ml microcentrifuge tubes
Steps:
- Measure out 2 ml of overnight culture into sterile 2-ml microcentrifuge tube (1).
- Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.
- Measure out 100 µl of <a href="#ice-cold GET buffer" >ice-cold GET buffer</a> into sterile 2-ml microcentrifuge tube (1).
Resuspend pellet by vortexing/by shaking vigorously. - Measure out 200 µl of 0.2M NaOH into sterile 2-ml microcentrifuge tube (2).
Add 2 mg of SDS.
Vortex the mixture for a few secs. - Measure out alkaline SDS solution into sterile 2-ml microcentrifuge tube (1).
Close the tube tightly and gently mix the contents by inverting the tube.
DO NOT VORTEX! The solution should become clear. - Add 150 µl of <a href="#ice-cold potassium acetate solution" >ice-cold potassium acetate solution</a>.
Close the tube tightly and gently mix the contents by inverting the tube.
DO NOT VORTEX! A precipitate should form. - Store the tube on ice for 3 - 5 mins.
- Centrifuge at maximum speed for 10 mins at room temperature and aspirate out 400 µl of top layer.
Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (3).
Discard bottom layer.
DO NOT PICK UP ANY PRECIPITATE!!! - Measure out 900 µl of 95% / 100% ethanol into sterile 2-ml microcentrifuge tube (4).
This is to precipitate the plasmid DNA. - Store at -80°C for 30 mins.
Use the -80°C freezer. - Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
- Add 1 ml of 70% EtOH.
Store at room temperature for 3 mins. - Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it.
Make sure the pellet is toward the outside. - Dry the pellet in air for 10 - 15 mins.
- Make sure the pellet is completely dry before this step.
Option 1: Add 20 µl of TE buffer.
(or)
Option 2: Add 20 µl of distilled water.Resuspend pellet by vortexing/by shaking vigorously.
The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.
(Optional)
Add 10 mg of <a href="#lysozyme" >lysozyme</a>.
Store at room temperature for 30 mins.
This step is essential for lysing gram-positive cells.
(Optional)
Measure out 400 µl of <a href="#PCA solution" >PCA solution</a> into sterile 2-ml microcentrifuge tube (3).
Close the tube tightly and gently mix the contents by inverting the tube.
Centrifuge at maximum speed for 3 mins at room temperature and aspirate out the top layer.
Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (4).
Discard bottom layer.
This helps remove any residual proteins.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 50 mins
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