Prather:Gibson CBA

Reagents

5x Isothermal Reaction Mix

3 ml 1 M Tris-Hcl (pH 7.5)

300 ul 1 M MgCl2

60 ul 100 mM dGTP

60 ul 100 mM dATP

60 ul 100 mM dTTP

60 ul 100 mM dCTP

300 ul 1 M DTT

1.5 g PEG-8000

300 ul 100 mM NAD

balance ddH2O

6 ml Total

Assembly Master Mix

320 ul 5X Isothermal Master Mix

0.64 ul 10 U/ul T5 exonuclease

20 ul 2 U/ul Phusion DNA Pol

0.16 ul 40 000 U/ul Taq DNA Ligase

860 ul ddH2O

1.2 ml Total

Aliquoted reaction and master mixes are stable at -20C and can withstand several freeze thaw cycles.

Protocol

1. PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments
2. Thaw assembly master mix and keep on ice until ready to be used
3. Mix 15 ul of assembly mixture with 5 ul total of cleaned PCR product (PCR Cleanup Kit or Gel Extraction) keeping DNA inserts in equimolar amounts
4. Incubate at 50 C for 15-60 min (60 min optimal).
5. Transform cells with no more than 1 ul of assembly mixture.

Comments

1. When preparing the isothermal reaction mix, add the PEG slowly to liquid. If added too quickly it will form a plug which will make mixing difficult (KS)
2. Have successfully used for a two way and three way ligation (KS)
3. There is a potential for point mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so so sequence to verify interfaces or leave spacers at the interfaces to 'absorb' these errors (KS)
4. I have used PCRs as is (with PCR cleanup only) and with gel extracted DNA. PCR Cleanup gives more colonies (more DNA, better quality (no agarose, QG contamination) but also has more false positives (PCR template plasmid). False positive could be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
5. I inadvertently designed my primers with 20 bp homology and a 20 bp spacer from the adjacent fragment and still got accurate plasmids. Possible to use shorter primers if desired (KS)

References

1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, and Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009 May;6(5):343-5. DOI:10.1038/nmeth.1318 |