Prather:Gibson CBA: Difference between revisions
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==Gibson Chew Back And Anneal Assembly: One Step Isothermal== | |||
''Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009.'' | ''Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009.'' | ||
===Reagents=== | |||
'''5x Isothermal Reaction Mix''' | '''5x Isothermal Reaction Mix''' <br /> | ||
3 ml 1 M Tris-Hcl (pH 7.5) | :3 ml 1 M Tris-Hcl (pH 7.5)<br /> | ||
300 ul 1 M MgCl2 | :300 ul 1 M MgCl2<br /> | ||
60 ul 100 mM dGTP | :60 ul 100 mM dGTP<br /> | ||
60 ul 100 mM dATP | :60 ul 100 mM dATP<br /> | ||
60 ul 100 mM dTTP | :60 ul 100 mM dTTP<br /> | ||
60 ul 100 mM dCTP | :60 ul 100 mM dCTP<br /> | ||
300 ul 1 M DTT | :300 ul 1 M DTT<br /> | ||
1.5 g PEG-8000 | :1.5 g PEG-8000<br /> | ||
300 ul 100 mM NAD | :300 ul 100 mM NAD<br /> | ||
:<ins>balance ddH2O</ins> <br /> | |||
:6 ml Total | |||
6 ml Total | |||
Assembly Master Mix | '''Assembly Master Mix'''<br /> | ||
320 ul 5X Isothermal Master Mix | :320 ul 5X Isothermal Master Mix | ||
0.64 ul 10 U/ul T5 exonuclease | :0.64 ul 10 U/ul T5 exonuclease | ||
20 ul 2 U/ul Phusion DNA Pol | :20 ul 2 U/ul Phusion DNA Pol | ||
0.16 ul 40 000 U/ul Taq DNA Ligase | :0.16 ul 40 000 U/ul Taq DNA Ligase | ||
860 ul ddH2O | :<ins>860 ul ddH2O</ins> | ||
:1.2 ml Total | |||
1.2 ml Total | |||
Aliquoted reaction and master mixes are stable at -20C and can withstand several freeze thaw cycles. | Aliquoted reaction and master mixes are stable at -20C and can withstand several freeze thaw cycles. | ||
Protocol | '''Protocol''' | ||
#PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments | |||
#Thaw assembly master mix and keep on ice until ready to be used | |||
#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 | |||
#Incubate at 50 C for 15-60 min (60 min optimal). | |||
#Transform cells with no more than 1 ul of assembly mixture. | |||
Revision as of 15:22, 8 August 2010
Gibson Chew Back And Anneal Assembly: One Step Isothermal
Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009.
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
- PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments
- Thaw assembly master mix and keep on ice until ready to be used
- 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
- Incubate at 50 C for 15-60 min (60 min optimal).
- Transform cells with no more than 1 ul of assembly mixture.
