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Team:NTU-Singapore/Notebook/11 June 2008
From 2008.igem.org
Wednesday 11 June
- Morning:
- Choon Kit, Hung:
- Gel electrophoresis for overnight digested products. Please refer to 10 June 2008 notebook
- Choon Kit, Hung:
(Reminder: the ratio of loading dye to test sample is 1:5)
Result:
+RBS 32 and 34 that were digested with XbaI and SpeI showed correct bands at 2kb. Control experiments (RBS 32 and 34 digested with EcoRI and PstI) also showed correct bands at 2kb.
+E7-1 PCR insert that was digested with XbaI and SpeI did not show any band, thus unsuccessful.
- Gel extraction of E7-0 insert (digested with XbaI and SpeI on Tuesday 10 June), RBS 32 and RBS 34 empty plasmid vectors (digested with XbaI and SpeI on Tuesday 11 June) using QIAgen Gel Extraction Kit.
- Ligation of E7-0 insert into RBS 32 empty plasmid vector.
| 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)
- 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)
| E7-1 PCR insert | |||||
| DNA | 16ul E7-0 insert + 2ul empty plasmid vector | ||||
| ligase buffer | 1ul T4 ligase buffer | ||||
| 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 |
| Total | 21 |
