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.