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

* 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