Team:NTU-Singapore/Notebook/10 June 2008

From 2008.igem.org

(Difference between revisions)
m (→Tuesday 10 June)
(→Tuesday 10 June)
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**Choon Kit, Hung: digestion of BBa_B0015 terminator and E7 plasmid with SpeI and XbaI. <br>
**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.
'''Objective''': to put E7 insert into empty vector from Terminator plasmid.
-
{|border="1"
+
{|style="color:white;background-color:black;" cellpadding="5" cellspacing="0" border="1"
|
|
|BBa_B0015 Terminator (vector w/o BBa_B0015 gene)
|BBa_B0015 Terminator (vector w/o BBa_B0015 gene)
Line 41: Line 41:
*Night (9 to 10 pm):
*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]
**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)
 +
{|style="color:white;background-color:black;" cellpadding="5" cellspacing="0" border="1"
 +
|
 +
|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
*Chin Chong & Darius

Revision as of 04:50, 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:
    • 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):
    • 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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