Team:NTU-Singapore/Notebook/25 June 2008

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=Wednesday 25 June=
=Wednesday 25 June=
-
*1030am: Hung: take out the agar plates incubated from Tuesday night. For details of the plasmids, please refer to [https://2008.igem.org/wiki/index.php?title=Team:NTU-Singapore/Notebook/24_June_2008 Tuesday notebook]  
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{|border="1" style="background-color:#ffffcc;" cellpadding="20"
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===Zhen Fu===
 +
*10am:  
 +
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
 +
===Chin Chong===
 +
*10am:
 +
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
 +
 
 +
 +
===Hung===
 +
*1030am:
 +
take out the agar plates incubated from Tuesday night. For details of the plasmids, please refer to [https://2008.igem.org/wiki/index.php?title=Team:NTU-Singapore/Notebook/24_June_2008 Tuesday notebook]  
**GFP reporters with standard, weak, medium and strong promoters all have colonies.
**GFP reporters with standard, weak, medium and strong promoters all have colonies.
**GFP reporter device BBa_E2040 however shows no colony.
**GFP reporter device BBa_E2040 however shows no colony.
Line 9: Line 38:
*11am: Group meeting to discuss about current obstacles encountered during wetlab.
*11am: Group meeting to discuss about current obstacles encountered during wetlab.
-
 
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===Choon Kit===
-
*12:15pm: Choon Kit: digestion for:
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*12:15pm:  
 +
digestion for:
**pFe with SpeI/PstI
**pFe with SpeI/PstI
**GFP with XbaI/PstI
**GFP with XbaI/PstI
**pFe with SpeI to check SpeI activity
**pFe with SpeI to check SpeI activity
**Terminator with SpeI to check SpeI activity<br>Incubate for 3 hours.
**Terminator with SpeI to check SpeI activity<br>Incubate for 3 hours.
 +
===Chin Chong, Zhen Fu===
 +
*2:30pm:
 +
Proceed with the characterization experiment
 +
#Cells that were left to grow in the morning reached OD 1.2
 +
#Centrifuge the cells at 4400 rpm and at 25 deg C
 +
#Remove the LB broth and Resuspend the cells with M9 medium
 +
#Introduce the cells into the 96-well
 +
#Induce the cells with varying concentrations of lactose (1-10mM) in 1mM increments
 +
#Did Control 1: Cells with water, Control 2: M9 medium, Control 3: Water
 +
#Place the 96 well into FLx800™ Fluorescence Microplate Reader and followed the protocol as before and measure RFU for 12 hrs
 +
 +
===Hung, Min-Lin===
 +
*3:30pm:
 +
Transform these following plasmids to Homemade top10 cells for amplification and obtaining new plasmids with higher purity (using new Miniprep Kit):
 +
**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.
 +
</div>

Latest revision as of 02:01, 28 October 2008

Contents

Wednesday 25 June

Zhen Fu

  • 10am:

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

Chin Chong

  • 10am:

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


Hung

  • 1030am:

take out the agar plates incubated from Tuesday night. For details of the plasmids, please refer to Tuesday notebook

    • 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

  • 12:15pm:

digestion for:

    • 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

  • 2:30pm:

Proceed with the characterization experiment

  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

  • 3:30pm:

Transform these following plasmids to Homemade top10 cells for amplification and obtaining new plasmids with higher purity (using new Miniprep Kit):

    • 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.
Retrieved from "http://2008.igem.org/Team:NTU-Singapore/Notebook/25_June_2008"