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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