Miniprep/GET buffer

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Cheap non-kit plasmid miniprep:
Cheap non-kit plasmid miniprep:
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# take 1.8 ml from an overnight culture into a 2 ml centrifuge tube, spin down 1 minute at full speed, and discard the medium
+
# Pipet 2 ml of overnight culture into a 2 ml centrifuge tube.
-
# resuspend the bacterial pellet by vortexing with 100 ul of chilled (4C) GET solution
+
# Centrifuge for 1 minute at maximum speed and discard the supernatant.
-
# lyse the bacteria by adding 200 ul of room temperature  alkaline SDS, not longer than 5 minutes.  Invert to mix: do not vortex. The solution should become clear.
+
# Add 100ul of refrigerated GET buffer to the pellet and vortex to resuspend.
-
# precipitate proteins, membranes, and chromosomal DNA with 150 ul chilled (4C) potassium acetate solution, mix by inverting gently.
+
# Add 2mg of SDS to 200ul of of room temperature 12mM NaOH and vortex to mix.  
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# centrifuge at full speed 5 to 10 min
+
# Apply the prepared alkaline SDS solution to the cell suspension and invert to mix. The solution should become clear. DO NOT VORTEX!!!
-
# transfer 400 ul of the clean supernatant into a new tube preloaded with 900 ul of 100% EtOH to precipitate the plasmid DNA
+
# Add 150ul of refrigerated potassium acetate solution, mix by inverting gently. The solution should form a precipitate.  DO NOT VORTEX!!!
-
# place the tubes in the -80 freezer for 30 minutes
+
# Centrifuge for 10 minutes at maximum speed.
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# centrifuge the precipitated plasmid DNA 10 to 15 minutes at full speed, and discard supernatant
+
# Carefully pipet 400ul of the clean supernatant into a new tube.  DO NOT PICK UP ANY PRECIPITATE!!!
-
# optional: wash the pellet with 70% EtOH, respin and discard the supernatant
+
# ADD 900ul of 100% (95% is ok too) EtOH to precipitate the plasmid DNA.
-
# air dry the pellet for 10-15 minutes at room temperature with the open tube on the bench
+
# Place the tubes in the -80 freezer for 30 minutes.
-
# resuspend the plasmid DNA in 20 ul of TE.  The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.
+
# Centrifuge the precipitated plasmid DNA 10 minutes at maximum speed and discard supernatant.
 +
# Carefully add 1ml 70% EtOH to the pellet and let sit for 3 minutes. 
 +
# Centrifuge at maximum speed for 3 minutes.  Make sure the pellet is toward the outside.
 +
# Discard the supernatant and air dry the pellet for 10-15 minutes.
 +
# 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.

Revision as of 15:49, 10 June 2009

Cheap non-kit plasmid miniprep:

  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 of room temperature 12mM NaOH and vortex to mix.
  5. Apply the prepared alkaline SDS solution to the cell suspension and invert to mix. The solution should become clear. DO NOT VORTEX!!!
  6. Add 150ul of refrigerated potassium acetate solution, mix by inverting gently. The solution should form a precipitate. DO NOT VORTEX!!!
  7. Centrifuge for 10 minutes at maximum speed.
  8. Carefully pipet 400ul of the clean supernatant into a new tube. DO NOT PICK UP ANY PRECIPITATE!!!
  9. ADD 900ul of 100% (95% is ok too) EtOH to precipitate the plasmid DNA.
  10. Place the tubes in the -80 freezer for 30 minutes.
  11. Centrifuge the precipitated plasmid DNA 10 minutes at maximum speed and discard supernatant.
  12. Carefully add 1ml 70% EtOH to the pellet and let sit for 3 minutes.
  13. Centrifuge at maximum speed for 3 minutes. Make sure the pellet is toward the outside.
  14. Discard the supernatant and air dry the pellet for 10-15 minutes.
  15. 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.


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

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