Prather:Gibson CBA

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(Gibson Chew Back And Anneal Assembly: One Step Isothermal)
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==Gibson Chew Back And Anneal Assembly:  One Step Isothermal==
==Gibson Chew Back And Anneal Assembly:  One Step Isothermal==
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Gibson Chew Back and Anneal Assembly (Gibson CBA) is a quick and easy method to construct plasmids without using restriction enzymes.  In this method, DNA fragments to be assembled are PCR amplified with 40 bp of overlap to the adjacent sequence (only one DNA fragment needs to have an overlap extension into the adjacent fragment).  These fragments are then mixed in a single pot with a single strand exonuclease to generate sticky ends and allowed to anneal before being repaired by a polymerase and a ligase.  In theory, many fragments can be assembled simultaneously (5 documented in the [[Prather:Gibson CBA#References|initial report]]) into a single product of up to ~100 kb+.  The method has several thermocycled variants of higher efficiency but the isothermal protocol offers almost comparable yields with greater simplicity.
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Gibson Chew Back and Anneal Assembly (Gibson CBA) is a quick and easy method to construct plasmids without using restriction enzymes.  In this method, DNA fragments to be assembled are PCR amplified with 40 bp of overlap to the adjacent sequence (only one DNA fragment per interface needs to have an overlap extension into the adjacent fragment).  These fragments are then mixed in a single pot with a single strand exonuclease to generate sticky ends and allowed to anneal before being repaired by a polymerase and a ligase.  In theory, many fragments can be assembled simultaneously (5 documented in the [[Prather:Gibson CBA#References|initial report]]) into a single product of up to ~100 kb+.  The method has several thermocycled variants of higher efficiency but the isothermal protocol offers almost comparable yields with greater simplicity.
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==Method==
==Method==
===Reagents===
===Reagents===

Revision as of 18:03, 8 August 2010

Contents

Gibson Chew Back And Anneal Assembly: One Step Isothermal

Gibson Chew Back and Anneal Assembly (Gibson CBA) is a quick and easy method to construct plasmids without using restriction enzymes. In this method, DNA fragments to be assembled are PCR amplified with 40 bp of overlap to the adjacent sequence (only one DNA fragment per interface needs to have an overlap extension into the adjacent fragment). These fragments are then mixed in a single pot with a single strand exonuclease to generate sticky ends and allowed to anneal before being repaired by a polymerase and a ligase. In theory, many fragments can be assembled simultaneously (5 documented in the initial report) into a single product of up to ~100 kb+. The method has several thermocycled variants of higher efficiency but the isothermal protocol offers almost comparable yields with greater simplicity.

Method

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. . pmid:19363495. PubMed HubMed [Gibson-nmeth-2009]

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