Gibson Assembly

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(New page: =Gibson Method, aka VLIC, aka one pot isothermal assembly= Original protocol from: [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html this paper], doi:10.1038/nmeth.1318 <br> ...)
(Specific Protocols)
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=Gibson Method, aka VLIC, aka one pot isothermal assembly=
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==Summary==
 +
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, 4 inserts + backbone, have been 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.
 +
 
 +
==Materials==
 +
*Master Mix
 +
**Store in -20C Freezer
 +
Prather Recipe
 +
'''5x Isothermal Reaction Mix''' <br />
 +
:3 ml    1 M Tris-Hcl (pH 7.5)<br />
 +
:300 μL  1 M MgCl2<br />
 +
:60 μL  100 mM dGTP<br />
 +
:60 μL 100 mM dATP<br />
 +
:60 μL 100 mM dTTP<br />
 +
:60 μL 100 mM dCTP<br />
 +
:300 μL 1 M DTT<br />
 +
:1.5 g PEG-8000<br />
 +
:300 μL 100 mM NAD<br />
 +
:<ins>balance  ddH2O</ins> <br />
 +
:6 ml Total
 +
 +
 
 +
'''Assembly Master Mix'''<br />
 +
:320 μL  5X Isothermal Master Mix
 +
:0.64 μL 10 U/μL  T5 exonuclease
 +
:20 μL  2 U/μL  Phusion DNA Pol
 +
:0.16 μL 40 000 U/μL Taq DNA Ligase
 +
:<ins>860 μL ddH2O</ins>
 +
:1.2 ml Total
 +
 
 +
[http://www.cambridgeigem.org/RFC57.pdf RFC57] Recipe
 +
'''5x isothermal reaction buffer:'''
 +
 
 +
* 25% PEG-8000
 +
* 500 mM Tris-HCl pH 7.5
 +
* 50 mM MgCl2,
 +
* 50mM DTT
 +
* 5mM NAD
 +
* 1mM each of the four dNTPs
 +
 
 +
'''1.33x Gibson Master Mix:'''
 +
 
 +
* Taq ligase (40u/ul): 50 ul
 +
* 5x isothermal buffer: 100 ul
 +
* T5 exonuclease (1u/ul): 2 ul
 +
* Phusion polymerase (2u/ul): 6.25 ul
 +
* Nuclease-free water: 216.75 ul
 +
 
 +
==Gibson Method, aka VLIC, aka one pot isothermal assembly==
Original protocol from: [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html this paper], doi:10.1038/nmeth.1318 <br>
Original protocol from: [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html this paper], doi:10.1038/nmeth.1318 <br>
-
(Shamelessly stolen and modified from [[JCA_OPIA | Chris's Version]]).<br>
 
-
Prepare a master mix, as detailed below.  Store them in 15 ul aliquots at -20 °C.  Then:
+
#PCR up your fragment of choice and gel purify
# Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
# Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
# Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
# Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
# Incubate at 50 °C for 15 to 60 min (60 min is optimal).
# Incubate at 50 °C for 15 to 60 min (60 min is optimal).
# Transform as usual
# Transform as usual
 +
 +
==Notes==
 +
*Ideally you have an overlap of 40 bp
 +
*Misc: - rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells. - Electrocompetence is ~ 1000x more effective so is tempting.
 +
*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)
 +
*The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies.  I've gone as high as 170 ng without any ill effects. (KS)
 +
*Have successfully used for a two way and three way ligation (KS)
 +
*There is a potential for mutations at the DNA boundaries which has yet to be quantified.  Paper suggests 1 every 50 assemblies or so.  Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)
 +
*I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies.  PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid).  False positives may be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
 +
*I once 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 less overlap if desired (KS)
 +
 +
==Specific Protocols==
 +
*[[Prather:Gibson_CBA]]
 +
*[[User:Andrew_Perry/Notebook/bbGibson/Gibson_assembly_protocol]]
 +
*[[Springer_Lab:_Isothermal_Assembly]]
 +
*[[Janet_B._Matsen:Guide_to_Gibson_Assembly|Guide to Gibson Assembly]]
 +
 +
==References==
 +
<biblio>
 +
#Gibson-nmeth-2009 pmid=19363495
 +
//Isothermal assembly method
 +
#Gibson-science-2008 pmid=18218864
 +
//Two step thermocycled assembly variant
 +
</biblio>
 +
[[Category:Protocol]]

Revision as of 18:07, 11 February 2014

Contents

Summary

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, 4 inserts + backbone, have been 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.

Materials

  • Master Mix
    • Store in -20C Freezer

Prather Recipe 5x Isothermal Reaction Mix

3 ml 1 M Tris-Hcl (pH 7.5)
300 μL 1 M MgCl2
60 μL 100 mM dGTP
60 μL 100 mM dATP
60 μL 100 mM dTTP
60 μL 100 mM dCTP
300 μL 1 M DTT
1.5 g PEG-8000
300 μL 100 mM NAD
balance ddH2O
6 ml Total


Assembly Master Mix

320 μL 5X Isothermal Master Mix
0.64 μL 10 U/μL T5 exonuclease
20 μL 2 U/μL Phusion DNA Pol
0.16 μL 40 000 U/μL Taq DNA Ligase
860 μL ddH2O
1.2 ml Total

RFC57 Recipe 5x isothermal reaction buffer:

  • 25% PEG-8000
  • 500 mM Tris-HCl pH 7.5
  • 50 mM MgCl2,
  • 50mM DTT
  • 5mM NAD
  • 1mM each of the four dNTPs

1.33x Gibson Master Mix:

  • Taq ligase (40u/ul): 50 ul
  • 5x isothermal buffer: 100 ul
  • T5 exonuclease (1u/ul): 2 ul
  • Phusion polymerase (2u/ul): 6.25 ul
  • Nuclease-free water: 216.75 ul

Gibson Method, aka VLIC, aka one pot isothermal assembly

Original protocol from: this paper, doi:10.1038/nmeth.1318

  1. PCR up your fragment of choice and gel purify
  2. Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
  3. Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
  4. Incubate at 50 °C for 15 to 60 min (60 min is optimal).
  5. Transform as usual

Notes

  • Ideally you have an overlap of 40 bp
  • Misc: - rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells. - Electrocompetence is ~ 1000x more effective so is tempting.
  • 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)
  • The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies. I've gone as high as 170 ng without any ill effects. (KS)
  • Have successfully used for a two way and three way ligation (KS)
  • There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)
  • I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies. PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid). False positives may be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
  • I once 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 less overlap if desired (KS)

Specific Protocols

References

  1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:19363495. PubMed HubMed [Gibson-nmeth-2009]
    Isothermal assembly method

  2. Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:18218864. PubMed HubMed [Gibson-science-2008]
    Two step thermocycled assembly variant

All Medline abstracts: PubMed HubMed
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