Jeff:LabNotes/Microbiome/2011-10-24

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Colony Prep[edit]

  • Purify with minelute

File:10-21-11 gel.jpeg

Ligation

  • I selected for 300-600 bp, so the mean size is ~450 bp. This corresponds to 32 ng DNA/rxn
  • Create the following: 32 ng DNA, 1 uL buffer, 1 uL vector, 1 uL t4 ligase, fill to 10 uL with water
  • Incubate at 16 C for 1 hr

Transfection

  • Add 1 uL of ligated product to thawed competent cells
  • Incubate on ice for 30 min
  • Heat shock at 42 C for 30 s
  • Put back on ice for 2 min
  • Add 250 uL SOC media to each and incubate at 37 C for 1 hr with shaking
  • Add 20 uL XGAL to each plate (warm up plates and XGAL to rt) and incubate in dark for 10 min
  • Add 50 uL cells to each plate and incubate overnight at 37 C