Team:Paris/6 August 2009

NoteBook
August 6th

 

Microscope
FM4-64 dying MG4 and Kayo delete Tol R protocol n°2 (05/08/09) but using PBS

->nearly the same as MgSO4

protocol n°2 (05/08/09)but 1ml of cells and 1µl of dye, one with MgSO4 and one with PBS

->not enough dye

Molecular Biology
Gel Migration
 * [Ladder 1kb|A1|nothing|A2|nothing|A3]using 1% agarose during 30min
 * A1 = Tg3p = 274bp (utiliser le ladder 100bp)
 * A2 = TE3 = 1036bp
 * A3 = ClyA (with RBS and poly G linker = 987bp




 * A1 and A2 didn't work we don't see it on the gel because it's not in E.Coli.
 * A3 didn't migrate very well
 * [Ladder 1kb|nothing|A5|nothing|A5|nothing] using 1% agarose during 30min
 * A5 = TolRII = 279bp



Purification and Digestion
 * Purification of A3, A4 directly in micro-column, A5, A6 using gel then micro-column
 * Digestion with XbaI and PSTI

New Miniprep Digestion by XbaI/PstI (XP) or XbaI/SpeI (XS) Digestion during 2h at 37°C. Gel migration
 * New Miniprep for P1: pSB2K3, P13: pSB1A3, P14: BBa_K136050 (RBS tetR)
 * Result: Just good for trash. Make new ON culture.
 * P1: pSB2K3 by XP
 * P2: BBa_J61002 (plasmid backbone with RFP) by XS
 * P7: Bba_R0040 (ptet) by XP
 * Gel migration for P1: pSB2K3, P2: BBa_J61002 (plasmid backbone with RFP), P7: BBa_R0040 (ptet)
 * [1kb|P1|P2|P7|100bp|/]



ON culture Transformation  
 * Result:
 * pSB2K3: wrong bande. Try again...
 * plasmid backbone with RFP: Wrong enzymes?!
 * ptet: good but the 1500bp band must not be there!
 * ON culture for S8: pSB2K3, S29: pSB1A3, S30: BBa_K136050 (RBS tetR) in order to done new miniprep
 * Resuspention of biobrick BBa_J2479 (1L7)
 * Transformation in competent DH5&alpha;