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Team:NTU-Singapore/Notebook/10 June 2008
From 2008.igem.org
Tuesday 10 June
- Morning:
- Choon Kit, Hung: digestion of [http://partsregistry.org/Part:BBa_B0015 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.
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.
- Gel electrophoresis for digested E7 (PCR product) and empty plasmid vector (extracted from BBa_B0015 plasmid)
- 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.
