Corum:T4 Ligation: Difference between revisions

Revision as of 15:19, 25 June 2012

Standard DNA ligation using NEB T4 ligase to form a vector.

For a 10 μL ligation reaction:

7 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be 10-20:1. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)
1 μL 10X ligation buffer
1 μL 10mg/ml BSA
0.5 μL T4 ligase

Add the reaction components together in order listed above. Mix gently and spin.
Incubate 16 °C overnight (8 hr minimum).

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

List troubleshooting tips here.
You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

Relevant papers, books, and websites

@@ Line 9: / Line 9: @@
 For a 10 μL ligation reaction:
-* 6.5 μL linker dsDNA
+* 7 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
-* 1 μL backbone dsDNA
+* 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be 10-20:1. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)
 * 1 μL 10X ligation buffer
 * 1 μL 10mg/ml BSA