Team:Paris/30 July 2009

Reception of strains

• New DH5α
• DH5α with g3p in plasmid

Glycerol stock of new DH5α [S6]

• Stock in 20% glycerol at 80°C

New competent DH5α by RbCl

• Competent bacteria
  • 1/50 ON culture of New DH5α
  • OD₆₀₀=0,6
• Transformation with puc19 (10pg/µl)
  • 250µl of RbCl competent DH5α
  • 12,5µl puc19
  • Leave 20 min in ice
  • 45s at 42°C
  • Leave 2 min in ice
  • Add 1ml of hot LB (42°C)
  • 1h at 37 under agitation
  • Put on plate (LB agar + Amp)

Overnight culture for [S3] Δnlp1, [S4] TB28, [S5] MG4, [S6] DH5α, [S7] DH5α g3p

• S3 Δnlp1: 5ml LB + 5µl Kan 1000x
• S4 TB28 : 5ml LB
• S5 MG4: 5ml LB + 5µl Kan 1000x + 250µl NaCl 20%
• S6 DH5α: 5ml LB
• S7 DH5α g3p: 6ml LB + 6µl Amp 1000x

Stock of NaCl 20%

• 100ml of H₂O
• 20g of NaCl
• Filtration