Protocols · Benchling

Materials

1. 5X isothermal (ISO) reaction buffer (25% PEG-8000, 500mM Tris-HCl pH 7.5, 50mM MgCl₂, 50mM DTT, 1mM each of the 4 dNTPs, and 5mM NAD). This is prepared as described below.
2. T5 exonuclease (Epicentre)

3. Phusion DNA polymerase (New England Biolabs)
4. Taq DNA ligase (New England Biolabs)
- Equipment 1. Heat block or thermocycler with PCR tubes.

Make Gibson Master Mixes

Prepare Iso Buffer

Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:3mL of 1M Tris-HCl pH 7.5150 μl of 2M MgCl₂₆₀ μl of 100mM dGTP 60 μl of 100mM dATP 60 μl of 100mM dTTP60 μl of 100mM dCTP300 μl of 1M DTT 1.5g PEG-8000300 μl of 100mM NADAdd water to 6mL Aliquot 100 μl and store at -20 °C₂. Prepare an assembly master mixture. This can be prepared by combining the following:

3mL of 1M Tris-HCl pH 7.5150 μl of 2M MgCl₂

60 μl of 100mM dGTP

60 μl of 100mM dCTP

60 μl of 100mM dTTP

60 μl of 100mM dATP

OR (alternatively 240µL of 100mM dNTPs to individual nucleotides*)

300 μl of 1M DTT

1.5g PEG-8000

300 μl of 100mM NAD

Add water to 6mL

Aliquot 100 μl and store at -20°C

Add Enzymes

Prepare an assembly master mixture. This can be prepared by combining the following:

320 μl 5X ISO buffer

Dilute 2µL of T5 exonuclease into 18µL of water*. Then add 6.4 μl of this now 1U/μl T5 exo (*dilution is recommended as pipetting 0.64µL is difficult and can result in more variation between mixes)

20 μl of 2U/μl Phusion pol

160 μl of 40U/μl Taq lig

Add water to 800µL* (The original protocol was in 1.2mL, but having more concentrated aliquots allows for more DNA to be added later).

Aliquot 10 μl* and store at -20°C. (*Alternatively you can aliquot 5µL and do assembly in a total volume of 10µL once DNA is added)

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 20-150bp overlaps. For DNA molecules overlapping by larger than 150bp, prepare the assembly mixture by using 3.2 μl of 10U/ μl T5 exo.

Add DNA and Perform Assembly

Thaw a 10* (*or 5) μl assembly mixture aliquot and keep on ice until ready to be used.

Add 10* (*or 5) μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100ng of each ~6kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250ng of each 150kb DNA segment).

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

If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli OR* 2.5µL into Zymo EZ-competent DH5alpha chemically competent E. coli (At the SGTC we routinely prepare our own chemically competent cells with this kit).