Gibson Assembly – DIY recipe

Published on Author Giorgio Gilestro

Reagents needed and providers:

Reagents
Tris Hcl pH 7.5 VWR A4263.0500 £21.90 500ml
PEG 8000 Sigma P1458-25ML £15.63 25ml
MgCl2 Thermofisher AM9530G £29.70 100ml
DTT VWR A3668.0050 £30.60 50ml
NAD NEB B9007S £16.80 0.2ml
dNTP Thermofisher 15363397 £49.50 1ml
Phusion NEB M0530S £56.00 100U
Taq Ligase NEB M0208S £49.60 2000U
T5 exonuclease NEB M0363S £41.60 1000U
Water VWR MDTH46-000-CI £33.00 6x100ml

2X ISO Reaction buffer

2X ISO Reaction buffer
Stock [] Final []
Tris Hcl pH 7.5 1M 200mM 220 1000
PEG 8000 40.00% 10.00% 275 1250
MgCl2 1M 20mM 22 100
DTT 1M 20mM 22 100
NAD 50mM 2mM 44 200
dNTP 10mM 0.4mM 44 200
H20 473 2150
total (ul) 1100 5000
Reactions # 104 474

All measurements are in ul. Make 133 ul aliquots and store at -20C. Good for one year. 1100 makes 8 aliquots, equivalent to about 104 final gibson reactions. Scale up using the attached XLS file (recipe.xls).

Master mix

Master mix
Stock [] Final []
2X ISO React. buff. 133.2 666
Phusion 2U/ul 0.033U/ul 3.3 16.5
Taq Ligase 40U/ul 5.3U/ul 26.6 133
T5 exonuclease* 5U/ul 0.005U/ul 0.2 1
H20 36.7 183.5
total (ul) 200 1000
Reactions # 13 66

Store at -20C. Good for 2 years. All measurements are in ul.

*T5 comes from NEB at 10U/ul. Make 1:2 dilution in IX T5 buffer (NEB #4) to create an intermediate stock solution

Credits and other protocols

There are several protocols for Gibson recipes on the internet. The one above is taken from the Kimble lab at UW-Madison (GibsonCloning.pdf). They usually work at ratio of 15ul of MIX to 5ul of DNA. The only one I found that works at 1:1 is the one by Ethan Ford (2x-gibson-assembly-master-mix.pdf)