"
Team:NTU-Singapore/Notebook/1 July 2008
From 2008.igem.org
Tuesday 1 July
- Hung, Darius, Choon Kit: try out ligation of pFe(insert) with GFP(vector); and pLacI(insert) with RBS34(vector):
- 1030-1130:prepare digestion mixtures for pFe,GFP,pLacI,RBS34. Use double digestion for the inserts and sequential digestion for the vectors.
- Vectors (1st digestion):incubate from 1130-230pm at 37 degrees C
- 1030-1130:prepare digestion mixtures for pFe,GFP,pLacI,RBS34. Use double digestion for the inserts and sequential digestion for the vectors.
| GFP | RBS34 | |
| DNA | 12.6 | 13.7 |
| Buffer2 | 2 | 2 |
| BSA | 0.2 | 0.2 |
| Mq water | 4.2 | 2.2 |
| XbaI | 2 | 2 |
| Total | 21 | 20 |
- Inserts (double digestion):incubate from 1130-130pm at 37 degrees C
| pLacI | pFe | |
| DNA | 27 | 16.5 |
| EcoRI Buffer | 2 | 2 |
| BSA | 0.2 | 0.2 |
| Mq water | 0 | 10 |
| EcoRI | 1 | 1 |
| SpeI | 1 | 1 |
| Total | 31 | 30 |
- 130pm-2pm: PCR purification (using PCR purification MinElute Kit) for pLacI,pFe to remove the enzymes (prevent star activity). After that, store the samples at 4 degrees fridge.
- 2pm-230pm: PCR purification (using PCR purification MinElute Kit) for GFP,RBS34 to remove the enzymes (prevent star activity)
- 230pm-6pm: 2nd digestion for GFP,RBS34 (including incubation for 3 hours). After this, we stored the samples at 4 degrees fridge.
