Team:Groningen/Protocols and Protocols/Gibsonassembly
Blue Bio Energy
Gibsonassembly
Protocol for Gibson assembly
ML-I
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.