Cheap miniprep

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

Using kit from Macherey-Nagel

  • Grow cells over night in 5ml liquid LB medium
  • Spin down cells from 2 ml in 2ml tube at 14000xg for 1 min
  • remove supernatant.
  • Repeat with another 2ml in the same tube
  • Add 150µl resuspension buffer and re-suspend all cells
  • Add 250µl lysis buffer (blue) and invert 5 time
  • Add 350µl neutralisation buffer and invert till the whole liquid is no longer blue
  • Spin down the cell debris and genomic DNA at 20.000xg 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 13000xg) 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 2min
  • Spin down (1min at 13000xg)
  • Add 30µl elution buffer AE
  • Incubate for 2min
  • Spin down (1min at 13000xg)
  • Label tube and trash column

Mini-prep of plasmids from E. coli

This is a very cheap and rather efficient miniprep method to extract plasmids from E. coli

  • Pick some colonies from the LB plate and grow each in a well (96 deep-well plate) with 0.5 ml LB+Amp (75 μg/ml) and incubate overnight at 37°C.
  • Transfer 5 μl of each well to a fresh U-bottom plate with 100 μl LB+Amp and place at 37°C.
  • With the remaining ~0.5 ml perform a miniprep.
  • Centrifuge cells down by spinning plate at 2250x g.
  • Pour of the LB and hit the plate upside down on a tissue to remove left over.
  • Resuspend cells in 100μl Resuspension buffer (solution #1). Use multichannel when using deepwell plate.
  • Add 100μl freshly made Lysis buffer (0.2 N NaOH, 1% SDS; solution #2) and mix.
  • Incubate for 5min.
  • Add 100μl Neutralisation Buffer (solution #3), mix well and incubate on ice for 5 min.
  • Transfer to a 2ml tube, this can be done during the 5 min incubation time.
  • Spin down pellet at 12,000 rpm for 5 min.
  • Remove the pellet with a toothpick if possible, else, transfer supernatant to fresh tube.
  • Add 700 μl 100% ethanol mix and leave at roomtemp for 5 min.
  • Spin down DNA at 15,000 rpm for 5min.
  • Pour out the supernatant and hit tube on tissue again to remove leftover liquid.
  • Rinse the pelleted DNA with 70% -20°C ethanol (in freezer 7).
  • Leave the tubes to dry completely
  • Add 20μl water to dissolve.

Resuspension buffer

Stock solution Final Concentration for 50ml
3.3M Glucose 50mM 0.76ml
1M Tris-HCl pH8.0 25 mM 1.25ml
0.5M EDTA pH8.0 10mM 1 ml
H2O 47 ml

Lysis buffer

Stock solution Final Concentration for 10ml for 5ml
10% SDS 1% SDS 1.0 ml 0.5 ml
1.0N NaOH 0.2N NaOH 2.0 ml 1.0 ml
H2O 7 ml 3.5 ml

Neutralization Buffer

Stock solution Final Concentration for 50ml
5M Potassium Acetate ~5M Potassium Acetate 44 ml
100% Glacial Acetic Acid 12% GAA 6 ml