Smolke:Protocols/Plasmid prep

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  • Decant your cultures into 1.5mL Eppendorf tubes to pellet the cells. This saves the cost of a 15mL Falcon tube and/or a 5mL pipet. -Josh
  • Follow Qiagen's QiaPrep Miniprep protocol, but skip step 7 (the Buffer PB wash).
  • Aliquot buffers from working stocks prepared by Andy (stored on bottom shelf of chemical cabinet).
    • When you aliquot from the stocks please use careful, sterile technique (even if you don't use sterile technique on your own stocks).
    • Take aliquots of ~15 ml of P1, P2, N3 in Falcon tubes and keep on your bench.
    • PE is made as a 5x stock (specifically, ethanol is not added). So, take an aliquot and add 4 volumes of EtOH to it (i.e., 3 ml stock + 12 ml EtOH). Make this up in 10-15 ml (final 1x volume) and make a new stock each month.
      • The worry with PE is that the ethanol will evaporate, so the total ethanol concentration will decrease over time. Keep your bottles tightly capped, and close them whenever you're not using it. I routinely use PE for months without any obvious problems. -Josh
    • Make aliquots of EB in 1-2 ml.