Team:Paris/5 August 2009

NoteBook
August 5th

 

Microscope
FM4-64 dying with Kayo protocol like n°2 = all cell fluo, no vesicle

protocol like n°6 = some cell fluo, noise, no vesicle

Molecular biology
Glycerol Stock
 * S20 JW4251, S21 JN0729, S22 JN0728, S23 JN0727, S24 JN0940, S24 JN0940, S25 JW5100, S25 JW5100, S26 JN5181, S27 FF64, S28 NEC280.

Miniprep
 * Miniprep for P1/2/3/4/5/7/8
 * Same protocol as yesterday. Storage at -20°C.

Purification
 * Purification (using 1%agarose at 30min migration) of P2=BBa-J61002 with XbaI PstI (put pTet forward).
 * D6=2053pb=P2 Digested
 * P2/nothing/ladder 100pb]




 * P2 purification checking with 1%agarose at 35 min at 75mV
 * P2



 PCR Gel migration
 * The purification is ok but don't use because it's doesn't have pTet, so we have to cut P2 with EcorI and SpeI next time.
 * Dilution oligo 1/10 (100µM to 10µM)
 * 2µl Oligo
 * 18µL H20 RNAse/DNAse free (Gibco)
 * Dilution DNA 1/10
 * 2µl Genomic DNA of K12 MG1655 &Delta;recA::Kan
 * 18µL H20 RNAse/DNAse free (Gibco)
 * Mix x1 Vf=50µl in PCR tube (in ice) (Add with this order)
 * 32,5µl H20 RNAse/DNAse free (Gibco)
 * 10µl Buffer Polymerase fusion 5x
 * 1µl dNTP
 * 2,5µl Oligo F 10µM
 * 2,5µl Oligo R 10µM
 * 1µl Genomic DNA of K12 1/10
 * 0,5µl Polymerase Fusion
 * Mix x6,5
 * 211,25µl H20 RNAse/DNAse free (Gibco)
 * 65µl Buffer Polymerase fusion 5x
 * 6,5µl dNTP
 * 6,5µl Genomic DNA of K12 1/10
 * Put 44,5µl of Mix x6,5 in a PCR tube
 * Add 2,5µl Oligo F 10µM
 * Add 2,5µl Oligo R 10µM
 * Add 0,5µl Polymerase Fusion
 * For A1/2/3/4/5/6, Program PHUSION:
 * 1: 98°C, 1min
 * 2: 98°C, 10sec
 * 3: 65°C, 30sec
 * 4: 72°C, 1min
 * Goto 2 29x
 * 72°C, 10min
 * 4°C, -
 * Gel 1%: 5µl of [A1|A2|100bp|A3|A4|1kb|A5|A6]
 * A1 = Amplification of N-term domain of M13's g3p = 274bp (Useless because we don't use g3p plasmide as matrice => Ctrl-)
 * A2 = Amplification of N-term domain of colicin E3 = 1036bp (Useless because we don't use the right matrice => Ctrl-)
 * A3 = Amplification of clyA with RBS in the primer = 987bp
 * A4 = Amplification of OmpA signal for export to periplasm = 135bp
 * A5 = Amplification of tolR = 279bp (Try again because of the second band)
 * A6 = Amplification of tatA with RBS = 1634bp (Try again because of the second band)

ON culture  
 * S8 DH5α(pSB2K3): LB Kan
 * S29 DH5α(pSB1A3): LB Amp
 * S30 S113 iGEM08 = DH5α(BBa_K136050): LB Amp