Team:Paris/31 July 2009

We don't see florescence

->do it again but wash the cells before the DID

Miniprep (Pure Yield Tm Plasmid Miniprep System) for [S7] which contain g3p expression plasmid [Protocol]

• S4 TB28 LB
• S5 MG4 LB Kan NaCl 1%
• S3 Δnlp1 LB Kan
• S8 DH5α with puc19 LB Amp (control for Amp resistance)

• 1/6I named 1I6 (BBa_R0040, TetR repressible promoter). Resistant to Ampicilin.
• 1/14N named 1N14 (BBa_10500, pBad/araC). Resistant to Kanamycin.
• 1/2J named 1J2 (BBa_J32015, PelB leader sequence). Resistant to Kanamycin.
• 1/18C named 1C18 (BBa_J61002, plasmid backbone). Resistant to Ampicilin.
• 1/7C named 1C7 (pSB2K3). Resistant to Kanamycin.
• 2/11P named 2P11 (BBa_I14032, pLac). Resistant to Kanamycin.
• 2/24C named 2C24 (BBa_B0014). Resistant to Kanamycin.
• 2/22P named 2p22 (BBa_K103006, OmpA linker). Resistant to Ampicilin.

• pFP141 (&DeltaN-Syn1A). Resistant to Kanamycin.
• pJM72 (His6-SNAP25). Resistant to Ampicilin.
• pTW2 (VAMP2-HIS6). Resistant to Ampicilin.

• 250µl of DH5α competent bacteria (by RbCl)
• Add 2µl of DNA (Biobrick)
• Incubation during 20min on ice
• Heat choc 45second at 42°C
• Leave on ice 2min
• Add 1ml of LB preheated at 42°C
• Incubation 1 hour at 37°C under agitation
• Put 100µl of culture under plate
• Centrifuge 2min at 6rpm MAX for 2 min
• Put 100µl of concentrated culture
• Incubate overnight at 37°C

• cfu/µg = nb colonies/µg DNA
• For RbCl, transformation efficiency = 1,2.10⁷cfu/µg