Difference between revisions of "Miniprep kit Macherey-Nagel"

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== Mini-prep of plasmids using kit (''E. coli'') ==
 
== Mini-prep of plasmids using kit (''E. coli'') ==
Using kit from Macherey-Nagel
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Using kit from Macherey-Nagel to perform a [[Miniprep]]
  
 
* Grow cells over night in 5ml liquid LB medium
 
* Grow cells over night in 5ml liquid LB medium

Revision as of 16:21, 3 January 2021

Mini-prep of plasmids using kit (E. coli)

Using kit from Macherey-Nagel to perform a Miniprep

  • Grow cells over night in 5ml liquid LB medium
  • Spin down cells from 2 ml in 2ml tube at 14000×g for 1 min
  • Remove supernatant.
  • Repeat with another 2ml in the same tube
    • Keep the remaining 1ml of cells for freezing if required
  • Add 150µl resuspension buffer and re-suspend all cells
  • Add 250µl lysis buffer (blue) and invert 5 times
  • Add 350µl neutralisation buffer and invert till the whole liquid is no longer blue
  • Spin down the cell debris and genomic DNA at 20.000×g for 3 min
  • Prepare the columns in tubes (or on the Vacuum manifold)
  • Add liquid without any pellet (better to add less supernatant than accidentally add gDNA)
  • Spin down (1min at 13000×g) (or vacuum) and remove flow through
  • Add 450µl washing solution and spin down (or vacuum)
  • Remove flow through and spin down to remove last bits of liquid
  • Move column to 1.5ml collection tube.
  • Add 30µl elution buffer AE
  • Incubate for 2 min
  • Spin down (1min at 13000×g)
  • Add 30µl elution buffer AE
  • Incubate for 2 min
  • Spin down (1min at 13000×g)
  • Label tube and trash column