Team:NTU-Singapore/Notebook/10 June 2008



  

=Tuesday 10 June= {|border="1" style="background-color:#ffffcc;" cellpadding="20"

Choon Kit, Hung
Objective: to put E7 insert into empty vector from Terminator plasmid. The amount added to each digestion sample is stated in the below table (unit: ul)
 * Morning:
 * Choon Kit, Hung: digestion of BBa_B0015 terminator and E7 plasmid with SpeI and XbaI.
 * 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 RBS B0032, RBS B0034

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.