Miniprep/GET buffer-PDF

Reagents

1. GET buffer = 50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8
2. Alkaline Buffer= 0.2 N NaOH
3. Potassium acetate = 3 M potassium acetate, 1.8 M acetic acid, no pH adjustment
4. SDS
5. PCA solution (optional) = 50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol
6. Lysozyme (optional)

Procedure

1. Pipet 2 ml of overnight culture into a 2 ml centrifuge tube.
2. Centrifuge for 1 minute at maximum speed and discard the supernatant.
3. Add 100ul of refrigerated GET buffer to the pellet and vortex to resuspend.
4. Freshly prepare the alkaline SDS solution by adding 10mg SDS per ml of 0.2M NaOH Solution.
5. Add 200μl of alkaline SDS solution to the cell suspension and invert to mix. DO NOT VORTEX! The solution should become clear.
6. Add 150ul of refrigerated potassium acetate solution and invert gently to mix. DO NOT VORTEX! A precipitate should form.
7. Store the tube on ice for 3-5 minutes.
8. Centrifuge for 10 minutes at maximum speed.
9. Carefully pipet 400ul of the clean supernatant into a new tube. DO NOT PICK UP ANY PRECIPITATE!!!
10. Add 900ul of 100% (95% is ok too) EtOH to precipitate the plasmid DNA.
11. Place the tubes in the -80 freezer for 30 minutes.
12. Centrifuge the precipitated plasmid DNA 15 minutes at maximum speed (cold if possible)and discard supernatant.
13. Carefully add 1ml 70% EtOH to the pellet and let sit for 3 minutes.
14. Centrifuge at maximum speed for 3 minutes. Make sure the pellet is toward the outside.
15. Discard the supernatant and air dry the pellet for 10-15 minutes.
16. Once the pellet is COMPLETELY DRY resuspend the plasmid DNA in 20 ul of TE or distilled water. The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.

Optional Steps

• After step 3 add 10mg lysozyme and incubate for 30 mins before proceeding with step 5. This step is essential for lysing gram-positive cells.
• After step 9 add 400µl PCA solution to the tube, invert to mix, and centrifuge for 3 minutes at maximum speed. Collect the upper phase by pipetting into a new tube and proceed with step 10. This step helps remove any residual proteins.

Notes

Scale-up

Even higher concentrations of plasmid have been obtained by scaling up this protocol 5x in 15ml centrifuge tubes. However, to do this a larger (i.e. slower) centrifuge was necessary to accomodate the larger tubes. The adjusted centrifugation steps were as follows:

Step 9: Centrifuge at 5500g (Max speed for this larger centrifuge) for 15 minutes.

Step 13: Centrifuge at 5500g for 20 minutes.