Team:NTU-Singapore/Notebook/3 July 2008



   =Thursday 3 July= {|border="1" style="background-color:#ffffcc;" cellpadding="20"

Chin Chong & Zhen Fu

 * Carried out characterization of Standard, Low, Medium and High Promoters that were sent to us in the Newsletter Vol. 1
 * 1) Cells that were grown overnight were diluted and grown to OD 1.2
 * 2) Cells were then centrifuge and the cell pellets were re-suspended with M9 medium with glycerol
 * 3) Each cell (promoter) will have two rows of wells assigned to it
 * 4) Pipette 200 uL of cells into the respective wells and add 50ul of water to each well
 * 5) Take the first sample at time zero and measure subsequent ones at 30 mins intervals using the plate reader at 37 deg C
 * 6) Stop the plate reader when extracting the cells from the well, taking note of the RFU value and recording it down on te eppendorf tube containing the extracted cells
 * 7) At every 30 mins, take 2 samples from each cell type (the 4 promoters)
 * 8) Centrifuge the cells at 4400 rpm, 25 deg C and for 5 mins. Remove the supernantant and store the cell pellets at -20 deg C
 * 9) Repeat this for the next 4 hours, collecting 8 sets of data
 * One eppendorh tube of each type of cells from the above experiment after the 4 hrs duration, were used as samples to be seen under the fluorecemce microscope
 * Cells fluorescene intensity was directly linked to the strenght of the promoters. The stronger the promoters, the brighter the fluorescene appears to be
 * Pictures and some movie clips were taken


 * E7+Imm successfully PCRed
 * Optimizing PCR reaction
 * Due to Self annealing primers(Primers-dimer), Increase Tm value and using 1ul of primers each instead of 2ul
 * Staining of gel
 * Unsuccessful initially with gel-star stain, however after post soaking of gel in stain buffer for 4hrs, bands became clearer and and missing bands reappeared.
 * New protocol for gel Runs--Post soak of gel in buffer for at least 30 mins

Choon Kit, Hung, Lu Chao:

 * Ligation of LacI-RBS34 and Fe-GFP again.