Miniprep/GET buffer: Difference between revisions

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==Notes==
==Notes==
*Concentrations of the reagents are as follows:
#GET buffer = 50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl  pH 8
#Alkaline SDS = 0.2 N NaOH, 1% SDS
#Potassium acetate = 3 M potassium acetate, 1.8 M acetic acid, no pH adjustment
#PCA solution = 50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol
*If using gram-positive cells: between steps 3 and 4 add 10mg of lysozyme and incubate for 30 minutes at 37°C.


==References==
==References==

Revision as of 11:18, 29 June 2009

A cheap, easy, and effective plasmid miniprep

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. Add 2mg of SDS to 200ul of room temperature 0.2M NaOH and vortex to mix.
  5. Apply the 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 10 minutes at maximum speed 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 4. This step is essential for lysing gram-positive cells.
  • After step 8 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 9. This step helps remove any residual proteins.

Notes

References

This protocol is based on a protocol described by Sambrook and Russell in their manual "Molecular Cloning" (2001) p:1.32-1.34

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