Team:Paris/16 August 2009

NoteBook
August 16th  

Lab work
Yesterday transformations results Miniprep Glycerol Stock Digestion Gel migration ON culture PCR screening Gel migration
 * g3p ON ligation at 16°C
 * Negative control concentrated = 0 bacteria
 * 1:1 concentrated = 1 bacteria
 * 1:7 concentrated = 1 bacteria
 * ClyA ON ligation at 16°C
 * Negative control concentrated = 0 bacteria
 * 1:1 rapport = 0 bacteria
 * 1:7 rapport = 0 bacteria
 * 1:1 concentrated = 10 bacteria
 * 1:7 concentrated = 0 bacteria
 * For the 6 positives clone of pFecA
 * For clone n°1 and n°2
 * 333ul of Glycerol 60%
 * 666ul of ON culture
 * Mix and store at -80°C
 * Digestion for the 6 clones of pFecA ligation, by XbaI and PstI
 * Mix
 * DNA (Miniprep) : 5ul
 * XbaI : 1ul (I think it's too much...but I think to this after doing it)
 * PstI : 1ul
 * BSA 100x : 0,2ul
 * Buffer NEB2 10x : 2ul
 * H2O : 9ul
 * 2h at 37°C
 * 1,5% agarose gel
 * 5ul drop
 * Good profil, expected band at 130bp. Maybe not enough digest bu we clearly see the 130bp band corresponding to the insert (pFecA). The 2000bp band correspond to pSB1A3
 * DH5&alpha; in order to done more competent bacteria (the stock is shrinking)
 * 4 g3p clones (7ml + 7ul Amp)
 * Same protocol as yesterday
 * Screening of 4 clones (maybe I invert the n°2 and the n°4)
 * g3p is around 1450bp... Second confirmation tomorrow but it seems very good!