Ligation

Materials:

• MQ water
• 1.5ml tubes
• 10x T4 Ligation buffer
• T4 Ligase
• Insert
• Vector

Reaction:

Component	20µl	Final concentration
MilliQ water	up to 20µl
10 ligation buffer	2µl	1x
Vector	xµl	20-100ng
Insert	xµl	3:1 molar ratio over vector
T4 DNA ligase 5U/µl	2µl	0.5U/µl

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