Team:Paris/15 August 2009

NoteBook
August 15th

 

Lab work
Transformation Miniprep Glycerol Stock Yesterday transformations results PCR screening protocol PCR screening Gel migration of the PCR screening
 * For overnight (at 16°C) ligation solution of g3p in pSB1A3
 * For overnight (at 16°C) ligation solution of ClyA in pSB1A3
 * ptet
 * pBad/araC
 * Double terminater
 * New strains
 * [S35] AA93 &Delta;fecA
 * [S36] fecIR mutant
 * [S37] fecIR wt
 * [S38] fecA
 * tatABC
 * No bacteria
 * pFecA
 * Negative control = 0 bacteria
 * 1:1 raport = 43 bacteria
 * 1:7 raport = 2 bacteria
 * 1:7 concentrated = Too much
 * g3p
 * Negative control = 0 bacteria
 * 1:1 raport = 1 bacteria
 * 1:7 raport = 0 bacteria
 * 1:7 concentrated = 16 bacteria
 * Program named COLONIES, use with VF2 and VR oligos
 * Lid 105°C
 * 1: 99°C 2min
 * 2: Sound
 * 3: Pause until press "Enter" button
 * 4: 95°C 5min
 * 5: 95°C 30sec
 * 6: 55°C 30sec
 * 7: 72°C [45sec] (1kb/bp, caution the universal oligo add 156bp)
 * 8: Goto 5 29x
 * 9:72°C 7min
 * 10: Hold at 4°C
 * Preparation of the ON culture (for Glycerol stock and Miniprep of the positives clones)
 * 7ml of LB+ Amp (because we use pSB1A3 as vector)
 * Bacterial solution
 * 100uL of H2O and few bacteria in a PCR tube
 * Put the cone in LB solution for ON culture
 * Put the PR tube in PCR and run the fist part of the program
 * PCR mix
 * Master Mix (2x) : 12,5ul
 * Oligo VF2 (10uM) : 0,5ul
 * Oligo VR (10uM) : 0,5ul
 * H2O : 6,5ul
 * Bacteria hoted at 99°C by PCR program : 5ul
 * Put PCR tube withe the PCR mix in the PCR and run the second part of the program (just push the "Enter" button)
 * For 6 clones of pFecA ligation in pSB1A3
 * Negative control without bacteria
 * "Positive" control with pSB1A3 wich have the mRFP (1050bp)
 * All the clone have se same profil (more than 300bp and under 400bp) and it seem to be good...Confirmation tomorow by enzymes digestion.
 * Negative control is...negative
 * Positive control is...positif but have a second band. (Must be confirmed)