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Team:University of Ottawa/1 August 2008
From 2008.igem.org
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:<li> No visible difference between: 5+ and 5-; and 15+ and 15-. 10- matches 1 hr better than 10+, which showed incomplete digestion. All samples other than 1 hr showed varying degrees of incomplete digestion. | :<li> No visible difference between: 5+ and 5-; and 15+ and 15-. 10- matches 1 hr better than 10+, which showed incomplete digestion. All samples other than 1 hr showed varying degrees of incomplete digestion. | ||
:<li> Microwave protocol is therefore not useful for time frames tested. However, current digestion processes can be shortened from 1 hour to 15 minutes. | :<li> Microwave protocol is therefore not useful for time frames tested. However, current digestion processes can be shortened from 1 hour to 15 minutes. | ||
| + | |||
| + | '''Digestion of Constructs OA, OB and T Amp Product''' | ||
| + | |||
| + | <TABLE BORDER=2> | ||
| + | <TR> <TD>Component</TD><TD>Volume(μL)</TD></TR> | ||
| + | <TR> <TD>Buffer3</TD><TD>1</TD></TR> | ||
| + | <TR> <TD>BSA</TD><TD>1</TD></TR> | ||
| + | <TR> <TD>Water</TD><TD>5</TD></TR> | ||
| + | <TR> <TD>SphI</TD><TD>1</TD></TR> | ||
| + | <TR> <TD>PstI</TD><TD>1</TD></TR> | ||
| + | <TR> <TD>DNA</TD><TD>3(1 μL each)</TD></TR> | ||
| + | </TABLE> | ||
| + | |||
| + | :<li> Deactivated at 80°C for 20 minutes | ||
| + | :<li> Added 1μL ligase and 1μL ATP to tubes. | ||
| + | :<li> Left one sample over the weekend to ligate, another for 5 seconds in the microwave, then to the 4°C fridge, and one for 10 seconds in the microwave then to the 4°C fridge. | ||
Revision as of 18:39, 7 August 2008
Today in the Lab
Chris
Digestion Experiment
| Sample | Condition |
| 5+ | 5 sec. in microwave |
| 5- | 5 sec. room temperature |
| 10+ | 10 sec. in microwave |
| 10- | 10 sec. room temperature |
| 15+ | 15 sec. in microwave |
| 15- | 15 sec. room temperature |
| 1 hr | 1 hour at 37 C |
| W+ | Water control microwaved for 10 sec. |
| W- | Water control at room temperature |
| Component | 1xvol.(μL) | 9xvol.(μL) |
| Buffer 3 | 1 | 9 |
| BglI | 1 | 9 |
| Water | 7 | 63 |
| DNA | 1 | 9 |
- All samples inactivated at 65°C for 20 minutes.
- Ran the products on a 1% gel at 80V for 40 minutes.
- No visible difference between: 5+ and 5-; and 15+ and 15-. 10- matches 1 hr better than 10+, which showed incomplete digestion. All samples other than 1 hr showed varying degrees of incomplete digestion.
- Microwave protocol is therefore not useful for time frames tested. However, current digestion processes can be shortened from 1 hour to 15 minutes.
Digestion of Constructs OA, OB and T Amp Product
| Component | Volume(μL) |
| Buffer3 | 1 |
| BSA | 1 |
| Water | 5 |
| SphI | 1 |
| PstI | 1 |
| DNA | 3(1 μL each) |
- Deactivated at 80°C for 20 minutes
- Added 1μL ligase and 1μL ATP to tubes.
- Left one sample over the weekend to ligate, another for 5 seconds in the microwave, then to the 4°C fridge, and one for 10 seconds in the microwave then to the 4°C fridge.
