Team:UC Davis/Protein Engineering Gibson

Gibson Cloning Protocol
Adopted from: Gibson et al. Nature Methods v. 6 n. 5; 343 (2009)

DNA NEEDED

1. Linearized dsVector
   *This can be generated through restriction digests or PCR amplification of the backbone
   

2. Linear dsDNA Inserts (1-10 chunks)
   *Each piece needs to have both a 5’ and 3’ overlap with the subsequent piece in the assembly
   *The overlaps should have a Tm of 65-75 (generally 20-40 base pairs)
   *If overlaps have polyG (>4) missanealing can occur and the reactions may fail

PREPARE STOCK SOLUTIONS:
Prepare 5X ISO buffer (25% PEG-8000, 500 mM Tris-HCl pH 7.5, 50 mM MgCl2, 50 mM DTT, 1 mM each of the 4 dNTPs, and 5 mM NAD). Six ml of this buffer can be prepared by combining the following:
3 ml of 1 M Tris-HCl pH 7.5
150 μl of 2 M MgCl2
60 μl of 100 mM dGTP
60 μl of 100 mM dATP
60 μl of 100 mM dTTP
60 μl of 100 mM dCTP
300 μl of 1 M DTT
1.5 g PEG-8000
300 μl of 100 mM NAD
Add water to 6 ml

Aliquot 100 μl and store at -20 °C

1. Prepare an assembly master mixture. This can be prepared by combining the following: 320 μl 5X ISO buffer 0.64 μl of 10 U/ μl T5 exo 20 μl of 2 U/μl Phusion polymerase 160 μl of 40 U/μl Taq ligase Add water to 1.2 ml

2. Aliquot 8 μl and store at -20 °C. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles. This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.

PERFORM REACTIONS:

1. Thaw a 8 μl assembly mixture aliquot and keep on ice until ready to be used.
   

2. Add 3 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
   

3. Incubate at 50 °C for 15 to 60 min (60 min is optimal).
   

4. If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli.