Template:Team:Groningen/CONTENT/PROTOCOLS/Gibsonassembly

Making 5X ISO buffer

5x ISO buffer was made according to the table below.

Content

Volume

Tris-HCl pH=7.5

3 mL

2M MgCl₂

150 μL

100mM dNTPs

60 μL

1M DDT

300 μL

PEG-8000

1.5 g

100mM NAD

300 μL

5x ISO buffer

Making enzyme mix for Gibson assembly

enzyme mix was made according to the table below.

Content

Volume

5x ISO buffer

20 μL

50 times diluted T5 exonuclease

2 μL

Phusion polymerase

1.25 μL

Taq ligase

10 μL

H₂O

41.75μL

Gibson assembly enzyme mix

Reaction

Add 5μL 10-100ng DNA in equimolar amount to 15μL enzyme mix.

Incubate for 1h at 50°C.