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This protocol is an adaptation of a standard bacterial alkaline lysis column-based miniprep kit. Presumably it will work with any vendor's kit

Materials Used:

  • YEPD Medium
  • Acid-washed glass beads 425-600 µm (Sigma, G8772-10G)
  • Reagents provided in Plasmid Miniprep Kit (buffers 1-3, wash buffer, and TE)

Day 0

  • Inoculate 10 ml YEPD or selective medium cultures with a patch of yeast on a yellow tip.
  • Grow overnight to saturation at 30°C and 220 rpm.

Day 1

  • Transfer 1.5 ml of the culture to a tube and centrifuge 2 minutes at 600 g. Remove the supernatant and repeat once (3 ml total).
  • Remove the supernatant. Add 250 µl buffer 1 as well as 250 µl glass beads (0.35 g) to each tube. Resuspend and beat on a mixer for 5 min.
  • Add 250 µl buffer 2, invert the tube 6-8 times and incubate a maximum of 5 min at room temperature.
  • Add 350 µl buffer 3, invert the tube 6-8 times.
  • Centrifuge 10 minutes at 13,000 rpm.
  • Apply the supernatant on the purification column and centrifuge 1 min.
  • Discard flow-through, add 750 µl wash buffer to the column and centrifuge 1 min.
  • Discard flow-through and centrifuge 2 min at 13.000 rpm.
  • Place the column on a clean 1.5 ml tube.
  • Add 30 µl TE preheated to 70°C, incubate 5 min, centrifuge 1 min at 13,000 rpm.