Nucleic acids purification

Plasmid Miniprep Protocol Start from 2-5 mL overnight culture for high copy vector or 5-10 mL for low copy vectors (volumes in brackets). This protocol is suitable for BAC DNA preparation prior to transformation into SW102 strain for recombineering experiments.

1) Resuspend pelleted bacterial cells in 250 µl (320 µL) Resuspension Buffer and transfer to a microcentrifuge tube. 2) Add 250 µl (320 µL) Lysis Buffer and mix by gentle inversion the tube 4–6 times to mix. Do not vortex, as this will result in shearing of genomic DNA 3) Add 350 µl (450 µL) Neutralization Buffer and invert the tube immediately but gently 4–6 times. 4) Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge. 5) Transfer the supernatant in a clean Eppendorf tube. 6) Add 0.6 volumes of isopropanol. 7) Centrifuge 10 minutes at maximum speed in a table top centrifuge 8) Wash with 70% ethanol. 9) Air dry and resuspend in 10 mM Tris pH = 7.4 (or ddH2O)

Resuspension Buffer Tris pH = 7.4 EDTA RNAse A

50 mM 10 mM 100 µg.mL-1

Lysis Buffer NaOH SDS

200 mM 1%

Neutralization Buffer KoAC

3M pH = 5.5

Nucleic Acid Purification 5