Jimenez-gomez lab:protocol miniprep without columns

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Institute Jean Pierre Bourgin
INRA - Versailles
Route de Saint Cyr
78026 Versailles Cedex
France
Tel: +33 1 30 83 33 16
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Miniprep with Promega reagents without using the columns


 

Day 1

  1. In a culture tube, inoculate 5ml of LB broth (with correspondent antibiotic) with a colony and incubate overnight at 37C with gentle shaking (about 60rpm).

 


 

Day 2

  1. Transfer 1.5ml of the overnight culture in a 1.5ml Eppendorf tube and centrifuge for 2 min at 5000rpm. (repeat once more with another 1.5 ml in the same tube). If desired, keep the remaining for a glycerol stock (see below)
  2. Discard the supernatant, and let the tube upside-down on a clean tissue (tela or similar) to completely remove the media
  3. Add 200μl of the cell Resuspension Solution and resuspend thoroughly by vortexing. IMPORTANT: from this point on, DON'T vortex or vigorously shake the samples!
  4. Add 200μl of the Cell Lysis Solution and immediately mix by inverting the tubes 4-5 times. Incubate until solution clears (approx. 1-5 min)
  5. Add 10μl of Alkaline Protease Solution and mix by inverting the tube 4-5 times. Incubate at room temperature for 5min (Do not incubate longer!)
  6. Add 280μl of the Neutralization Solution, immediately mix by inverting the tube 4-5 times
  7. Centrifuge for 5-10min at max speed
  8. Transfer the supernatant in a new 1.5ml Eppendorf tube and add 100% ethanol at -20°C until filled
  9. Incubate on ice for exactly 2min
  10. Centrifuge for 30min at 4C at max speed
  11. Discard the supernatant and wash the pellet with 500μl of 70% ethanol
  12. Air dry the pellet and resuspend in 100μl of TE (not for sequencing!!) or water.
  13. (optional) Add RNAsa to a final concentration of 10μg/ml (2μl enzyme (500μg/ml) in 100 μl buffer) and incubate at room temperature for 3 hours.


Glycerol stocks


  1. Add 300 μl Glycerol 50% to a 1.5 ml tube
  2. Add 700 μl of the cell culture
  3. Mix and freeze in liquid nitrogen
  4. Store at -80°C

 

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