Team:NTU-Singapore/Notebook/10 June 2008

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=Tuesday 10 June=
=Tuesday 10 June=
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<div class="quote" style="margin:20px;">
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{|border="1" style="background-color:#ffffcc;" cellpadding="20"
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|
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===Choon Kit, Hung===
*Morning:
*Morning:
-
**Choon Kit, Hung: digestion of BBa_B0015 terminator and E7 plasmid with SpeI and XbaI.
+
**Choon Kit, Hung: digestion of [http://partsregistry.org/Part:BBa_B0015 BBa_B0015 terminator] and E7 plasmid with SpeI and XbaI. <br>
-
{|border="1"
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'''Objective''': to put E7 insert into empty vector from Terminator plasmid.
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{|style="color:white;background-color:black;" cellpadding="5" cellspacing="0" border="1"
|
|
-
|BBa_B0015 Terminator
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|BBa_B0015 Terminator (vector w/o BBa_B0015 gene)
|E7
|E7
|-
|-
Line 34: Line 56:
|'''10 ul'''
|'''10 ul'''
|'''40 ul'''
|'''40 ul'''
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|}
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*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.
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**Inoculation for T7 promoter.
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*Night (9 to 10 pm):
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**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]
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The amount added to each digestion sample is stated in the below table (unit: ul)
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{|style="color:white;background-color:black;" cellpadding="5" cellspacing="0" border="1"
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|
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|RBS 32
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|RBS 34
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|RBS 32 (control)
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|RBS 34 (control)
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|E7-1 PCR insert
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|-
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|DNA
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|5
 +
|5
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|2
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|2
 +
|30
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|-
 +
|buffer
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|1ul buffer 2
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|1ul buffer 2
 +
|1ul EcoRI buffer
 +
|1ul EcoRI buffer
 +
|4ul buffer 2
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|-
 +
|Digestive enzyme
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|1ul SpeI + 1ul XbaI
 +
|1ul SpeI + 1ul XbaI
 +
|0.5ul EcoRI + 0.5 PstI
 +
|0.5ul EcoRI + 0.5 PstI
 +
|1ul SpeI + 1ul XbaI
 +
|-
 +
|BSA (1/10 of the buffer used)
 +
|0.1
 +
|0.1
 +
|0.1
 +
|0.1
 +
|0.4
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|-
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|DI water
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|1.9
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|1.9
 +
|5.9
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|5.9
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|3.6
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|-
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|Total
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|10
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|10
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|10
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|10
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|40
|}
|}
-
*Chin Chong & Darius
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===Chin Chong & Darius===
**Prepare new stock for Ecoli cells containing Laci-GFP
**Prepare new stock for Ecoli cells containing Laci-GFP
**Auto-clave the tips and LB broth
**Auto-clave the tips and LB broth

Latest revision as of 00:33, 28 October 2008


Tuesday 10 June

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.

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:
    • 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)

RBS 32 RBS 34 RBS 32 (control) RBS 34 (control) E7-1 PCR insert
DNA 5 5 2 2 30
buffer 1ul buffer 2 1ul buffer 2 1ul EcoRI buffer 1ul EcoRI buffer 4ul buffer 2
Digestive enzyme 1ul SpeI + 1ul XbaI 1ul SpeI + 1ul XbaI 0.5ul EcoRI + 0.5 PstI 0.5ul EcoRI + 0.5 PstI 1ul SpeI + 1ul XbaI
BSA (1/10 of the buffer used) 0.1 0.1 0.1 0.1 0.4
DI water 1.9 1.9 5.9 5.9 3.6
Total 10 10 10 10 40

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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