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Team:University of Ottawa/5 August 2008
From 2008.igem.org
Today in the Lab
Chris
- Inoculation control was contaminated; will reperform later today.
Digestion of Construct OA, OB and T Amp Product
- In order to increase the scope of our microwave digestion experiment, I added the following samples for comparison purposes.
| Sample | Condition |
| 1' | 5 sec. in microwave, 2 minutes on dry ice |
| 2' | 2x(5 sec. in/2 min. dry ice) |
| 3' | 10x(5 sec. in/2 min. dry ice) |
- Digestion was performed according to previous protocol, including denaturation at 80°C and addition of ligase+ ATP.
- Ran all products on a 1% gel; however, did not run digestion products as a control and had to redo the experiment.
Ligation Microwave Optimisation
| Component | 1xvol.(μL) | 6xvol.(μL) |
| Buffer3 | 1 | 6 |
| BSA | 1 | 6 |
| Water | 3 | 18 |
| PstI | 1 | 6 |
| SphI | 1 | 6 |
| DNA | 3(1 of each) |
- Incubated at 37°C for 15 minutes, followed by 20 minutes at 80°C to denature the enzymes
2. Ligation
- Added 1μL each ligase and ATP to the digestion samples before subjecting them to the following conditions:
| Sample | Condition |
| ON | Overnight at room temperature |
| 5x1 | 5 sec. in microwave |
| 5x1ICE | 5 sec. in microwave/30 sec. on dry ice |
| 5x10ICE | 5 sec. in/30 sec. ice ten times |
| 6 | Room temperature for six minutes |
| Water | Water control |
| DP | Digestion Products only |
- All samples stored at 4°C following ligation
- Also: inoculated 5 samples each of Matt's 3:1 and 6:1 PTP2/PSSA42 construct for minipreparation tomorrow.
