Choon Kit, Hung

• Morning:
  • Choon Kit, Hung: digestion of [http://partsregistry.org/Part:BBa_B0015 BBa_B0015 terminator] and E7 plasmid with SpeI and XbaI.
    

Objective: to put E7 insert into empty vector from Terminator plasmid.

• Afternoon:
  • Gel electrophoresis for digested E7 (PCR product) and empty plasmid vector (extracted from BBa_B0015 plasmid)
    Result: E7 insert showed a correct 2kb band. Empty plasmid vector showed a smear of bands, hence it was not digested properly by SpeI and XbaI.
  • Inoculation for T7 promoter.
• Night (9 to 10 pm):
  • Do an overnight digestion (cut with XbaI and SpeI) to obtain empty plasmid vector from [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 RBS B0032], [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS B0034]

The amount added to each digestion sample is stated in the below table (unit: ul)

Chin Chong & Darius

• • Prepare new stock for Ecoli cells containing Laci-GFP
  • Auto-clave the tips and LB broth
  • Re-run PCR for E7-imm to produce more stock for further use
  • Carry out LacI-GFP characterization with varying concentration using a fluorescence mutiplate reader (96 well)
    • 2 range of IPTG/lactose were investigated over 4 hours
    • 1st range 0-2 mM in 0.2mM increments
    • 2nd range 0-10 mM in 2mM increments
  • Results from the lacI-GFP characterization shows that there is an increasing trend in GFP fluorescence for all samples
  • Wells containing higher concentration of IPTG and lactose seems to have a higher fluorescence reading
  • Effective range of IPTG should be from 0-2 mM. As readings from 4 to 10 mM appears to be similar.