Team:NTU-Singapore/Notebook/25 June 2008



  

=Wednesday 25 June= {|border="1" style="background-color:#ffffcc;" cellpadding="20"

Zhen Fu
take out the 50ml tubes incuated from Tuesday evening. Prepare the varying lactose concentratons (1 to 10 mM) as well as the controls in 1.5ml eppendorh tubes as preperations for Characterization
 * 10am:

Chin Chong
Diluted the cells(Top 10 and BL21) from the 50ml tubes to OD 0.590 and re-incubate them to allow them to grow to 1.2
 * 10am:

Hung
take out the agar plates incubated from Tuesday night. For details of the plasmids, please refer to Tuesday notebook
 * 1030am:
 * GFP reporters with standard, weak, medium and strong promoters all have colonies.
 * GFP reporter device BBa_E2040 however shows no colony.
 * GFP producer BBa_E4080 shows a few colonies.
 * LacI-GFP shows no colony.
 * GFP self-ligation shows a few colonies.
 * pFe-GFP shows no colony.


 * 11am: Group meeting to discuss about current obstacles encountered during wetlab.

Choon Kit
digestion for:
 * 12:15pm:
 * pFe with SpeI/PstI
 * GFP with XbaI/PstI
 * pFe with SpeI to check SpeI activity
 * Terminator with SpeI to check SpeI activity Incubate for 3 hours.

Chin Chong, Zhen Fu
Proceed with the characterization experiment
 * 2:30pm:
 * 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
 * 3) Remove the LB broth and Resuspend the cells with M9 medium
 * 4) Introduce the cells into the 96-well
 * 5) Induce the cells with varying concentrations of lactose (1-10mM) in 1mM increments
 * 6) Did Control 1: Cells with water, Control 2: M9 medium, Control 3: Water
 * 7) Place the 96 well into FLx800™ Fluorescence Microplate Reader and followed the protocol as before and measure RFU for 12 hrs

Hung, Min-Lin
Transform these following plasmids to Homemade top10 cells for amplification and obtaining new plasmids with higher purity (using new Miniprep Kit):
 * 3:30pm:
 * pT7 (ampR)
 * LacI (ampR)
 * RBS 34 (ampR)
 * RBS 32 (ampR)
 * GFP E0240 (ampR)
 * Terminator (AK resistance)
 * pFe (ampR)
 * empty plasmid with AmpR

Chin Chong, Zhen Fu

 * 5pm: Prepare more empty 500ml bottles to autoclave for future use

Hung

 * 6pm: cell cloning on agar plates for the above plasmids
 * 6:30pm: inoculate in 5ul LBA each for:
 * LacI-GFP in top10
 * LacI-GFP in BL21 cell
 * self-ligated GFP cell
 * E0480 GFP producer cell

Choon Kit
3pm to 7pm:
 * Gel electrophoresis: controls of pFe and Terminator each show only 1 single band, which mean there's nothing wrong with SpeI.
 * Gel extraction of pFe (cut with SpeI and PstI) and GFP (cut with XbaI and PstI).
 * Gel electrophoresis again to check the extracted plasmids.