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Team:Groningen/protocols/Ligation
From 2013.igem.org
Ligation Reaction
Materials:
- MQ water
- 1.5 ml tubes
- 10x T4 Ligation buffer
- T4 Ligase
- Insert
- Vector
Reaction mixture:
| Component | 20 µl | Final concentration |
|---|---|---|
| MilliQ water | up to 20 µl | |
| 10 ligation buffer | 2 µl | 1x |
| Vector | x µl | 20-100 ng |
| Insert | x µl | 3:1 molar ratio over vector* |
| T4 DNA ligase 5 U/µl | 2 µl | 0.5 U/µl |
* When restricted PCR products are ligated together, a 1:1 molar ratio is used.
All the reagents are added following the order listed in the table above [1].
After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22°C) for 20 min.
[1] http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf
iGEM 2013 Groningen
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