Team:Groningen/protocols/Ligation

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 5U/µl	2 µl	0.5U/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.
After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22°C) for 20 min.
Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf