Single Insert Gibson Reaction Calculator

Please note that this is a generic protocol that assumes an enzyme that functions at 37C / is heat inactivable / uses buffers compatible with Gibson. Check the specifics of your enzyme before proceeding.

backbone concentration: ng/uL

backbone size: kB

insert concentration: ng/uL

insert size: bp

Reaction parameters

desired molar ratio insert to backbone

gibson mix volume final uL

Digestion mix

dH20
enzyme	1
10X buffer	1
backbone
TOTAL	10

After adding together all the components, incubate for 60 minutes in a heatblock set to 37C. Then heat inactivate the enzyme by incubating the mix at 65C for 20 minutes.

Gibson assembly mix

2x master mix	2.5
insert	1
digestion mix	1
dH20	1
TOTAL	5

After adding together all the components, incubate for 25 minutes in a heatblock set to 50C. Use all 5 uL for subsequently transforming 50 uL of competent bacteria.