Editing Gibson Assembly (section)

== Protocol 1: Gibson Assembly for Cloning PCR Fragment into a Vector ==

1) Prepare 6 ml of '''5X ISO Buffer '''in a 15 ml falcon tube as follows:

3 ml 1 M Tris-HCl pH 7.5

+ 150µl 2 M MgCl<sub>2</sub>

+ 240 µl 100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP)

+ 300µl 1 M DTT

+ 1.5 g PEG-8000

+ 300µl 100 mM NAD

+ dH20 to 6 ml

Store at -20 oC in 320 µl aliquots.

2) Prepare '''0.5&nbsp; ml''' of '''3.2 X Gibson Cloning master mix''' as follows:

320 µl 5X ISO Buffer

+ 0.64 µl 10 U/µl T5 exonuclease*

+ 20 µl 2 U/µl Phusion polymerase

+ 160 µl 40 U/µl Taq ligase

'''DO&nbsp;NOT&nbsp;ADD WATER'''

Store at -20 oC in '''5 µl '''aliquots.This is 3.2 X mix. This mix allows you to add sample and water to 16 µl

This is optimized for 20-150 bp sequence homology overlaps

3) Thaw a '''5 µl '''aliquot of 3.2X Gibson assembly master mix, and keep on ice until use.

4) Measure the DNA concentration (ng/µl) of each assembly piece.

5) Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces in '''11µl''' (e.g., elute DNA from spin column with 11 µl water after gel purification) to the thawed '''5µl''' 3.2X master mix in a '''16 µl''' total volume assembly reaction mixture as follows:

linearized vector backbone (100 ng) + each additional assembly piece (to equimolar with backbone)==11&nbsp; µl

+ 5 µl 3.2 X Gibson&nbsp;Cloning assembly master mix

6) Incubate the assembly reaction at '''50 oC '''for 15 ~ '''60 minutes''', and then place on ice.

7) Transform 5 ~15 µl of the assembly reaction into 100µl of competent E. coli and/or run an agarose gel to check for successful assembly.