Prather:Gibson CBA: Difference between revisions
(New page: Gibson Chew Back And Anneal Cloning: One Step Isothermal Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009. 5x Isothermal Reaction Mix 3 ml 1 M Tris-Hcl (pH 7.5) 300 ul 1 M MgC...) |
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Gibson Chew Back And Anneal Cloning: One Step Isothermal | Gibson Chew Back And Anneal Cloning: One Step Isothermal | ||
Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009. | [em]Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009.[/em] | ||
Revision as of 15:10, 8 August 2010
Gibson Chew Back And Anneal Cloning: One Step Isothermal [em]Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009.[/em]
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 1.85 mL 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.
