Team:Paris/31 July 2009

NoteBook
July 31th

 

Lab work
DID dying to DH5a and MG4 We don't see florescence

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

Miniprep Miniprep (Pure Yield Tm Plasmid Miniprep System) for [S7] which contain g3p expression plasmid [Protocol] Culture ON for glycerol stock Biobrick resuspention Transformation of precited Biobrick Transformation efficiency (cfu/µg)  
 * S4 TB28 LB
 * S5 MG4 LB Kan NaCl 1%
 * S3 &Delta;nlp1 LB Kan
 * S8 DH5&alpha; 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 DH5&alpha; 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