Difference between revisions of "Team:Michigan/Experiments"
Line 59: | Line 59: | ||
<h4>Ligation Protocol with T4 DNA Ligase</h4> | <h4>Ligation Protocol with T4 DNA Ligase</h4> | ||
+ | <br><br> | ||
+ | 1. Set up the following reaction in a microcentrifuge tube on ice.<br> | ||
+ | 2. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) <br> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/6/67/Experiment1.png" alt="Experiment 1" style="width:75%;height:75%;"> | ||
+ | * The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.<br><br> | ||
+ | 3. Gently mix the reaction by pipetting up and down and microfuge briefly.<br> | ||
+ | 4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.<br> | ||
+ | 5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours<br> | ||
+ | 6. Heat inactivate at 65°C for 10 minutes.<br> | ||
+ | 7. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.<br> | ||
+ | |||
</html> | </html> |
Revision as of 05:37, 20 November 2015
Experiments and Protocols
Ligation Protocol with T4 DNA Ligase
1. Set up the following reaction in a microcentrifuge tube on ice.
2. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.)
![Experiment 1](https://static.igem.org/mediawiki/2015/6/67/Experiment1.png)
3. Gently mix the reaction by pipetting up and down and microfuge briefly.
4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours
6. Heat inactivate at 65°C for 10 minutes.
7. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.