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	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>		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>
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−	<img src="https://static.igem.org/mediawiki/2015/6/67/Experiment1.png" alt="Experiment 1" style="width:75%;height:75%;">	+	<img src="https://static.igem.org/mediawiki/2015/6/67/Experiment1.png" alt="Experiment 1" style="width:75%;height:75%;"><br>
	* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.<br><br>		* 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>		3. Gently mix the reaction by pipetting up and down and microfuge briefly.<br>

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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.)


* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

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.