Team:NTU-Singapore/Notebook/27 June 2008



  

=Friday 27 June= {|border="1" style="background-color:#ffffcc;" cellpadding="20"
 * Hung: miniprep for pFe,Terminator,RBS32,RBS34,pT7,GFP,empty plasmid AmpR,LacI
 * Choon Kit: inoculate pFe-GFP
 * Choon Kit: inoculate pFe-GFP


 * Chin Chong, Zhen Fu: Proceed with the characterization experiment for Quantification of RFU
 * 1) Cells that were left to grow in the morning reached OD 1.2
 * 2) Centrifuge the cells at 4400 rpm and at 25 deg C for 5 minutes
 * 3) Remove the LB broth and Resuspend the cells with 5ml of M9 medium with glycerol
 * 4) Incubate at 37 deg C and grow the cells to OD 1.2
 * 5) Introduce the cells into the 96-well
 * 6) Induce the cells with varying concentrations of lactose (1-10mM) in 1mM increments
 * 7) Did Control 1: Cells with water, Control 2: M9 medium, Control 3: Water
 * 8) Place the 96 well into FLx800™ Fluorescence Microplate Reader and followed the protocol as before and measure RFU for 12 hrs
 * 9) Range of RFU interested was 10-70 RFU
 * 10) Data points of 5 RFU increments ranging from 10 to 70 RFU were noted when the cella are allowed to grow for 12 hrs.
 * 11) Whenever the desired RFU readings were measured, the Microplate Reader was paused and the desired well contents were extracted out into eppendrof tubes. For each desired RFU readings, two samples were extracted.
 * 12) The tubes were centrifuged at 4400 rpm, 25 deg C for 5 minutes
 * 13) The supernantant was removed carefully with a pippete so as to prevent removing any cell contents
 * 14) The tubes containing only the cell contents were then stored in -80 deg C fridge for further experiment (SDS PAGE) in future