Team:NTU-Singapore/Notebook/10 June 2008

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=Tuesday 10 June=
=Tuesday 10 June=
*Morning:
*Morning:
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**Choon Kit, Hung: digestion of BBa_B0015 terminator and E7 plasmid with SpeI and XbaI.
+
**Choon Kit, Hung: digestion of BBa_B0015 terminator and E7 plasmid with SpeI and XbaI. <br>
 +
'''Objective''': to put E7 insert into empty vector from Terminator plasmid.
{|border="1"
{|border="1"
|
|
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|BBa_B0015 Terminator
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|BBa_B0015 Terminator (vector w/o BBa_B0015 gene)
|E7
|E7
|-
|-
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|'''40 ul'''
|'''40 ul'''
|}
|}
 +
**Afternoon:
 +
**#Gel electrophoresis for digested E7 (PCR product) and empty plasmid vector (extracted from BBa_B0015 plasmid)<br>
 +
'''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.<br>
 +
**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]
 +
*Chin Chong & Darius
*Chin Chong & Darius

Revision as of 04:25, 13 June 2008

Tuesday 10 June

  • Morning:
    • Choon Kit, Hung: digestion of BBa_B0015 terminator and E7 plasmid with SpeI and XbaI.

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

BBa_B0015 Terminator (vector w/o BBa_B0015 gene) E7
DNA 5 30
Buffer 2 1 4
BSA 0.1 0.4
SpeI 1.5 1.5
XbaI 1.5 1.5
H2O 0.9 2.6
Total 10 ul 40 ul
    • Afternoon:
      1. 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.

    • Night (9 to 10 pm):
      1. 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]


  • 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.
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