Difference: ProtocolsGibsonCloning (13 vs. 14)

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Gibson Assembly

Background and Design

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Gibson_Assembly_pic.jpg Gibson Cloning is a technique of DNA construct assembly that allows one to join multiple linear segments into either one large linear segment or, if the segments contain the appropriate components and overlaps, an intact plasmid. This protocol follows the one-step ISO assembly of overlapping dsDNA protocol. In order to assemble segments of DNA via Gibson Cloning, they must contain at least 40bp of homology to the segment they are being joined to. For example, if one was to make a construct that was Seg1-Seg2-Seg3 from individual PCRs of Seg1, Seg2, and Seg3, the 3' end of Seg1 would need 40bp of homology to the 5' end of Seg2 and Seg3 would require its 5' end to have 40bp of homology to Seg2. For constructing a plasmid, use a linearized vector backbone as one of your segments.
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Gibson Cloning is a technique of DNA construct assembly that allows one to join multiple linear segments into either one large linear segment or, if the segments contain the appropriate components and overlaps, an intact plasmid.
 
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This protocol follows the one-step isothermal assembly of overlapping dsDNA. In order to assemble segments of DNA via Gibson Cloning, they usually must contain at least 20bp of homology to the segment they are being joined to (Tm of overlapping region must be >= 48C). Homology overlaps can vary in length from as few as 15bps up to 80bps -- efficacy depends on number of fragments assembled, as well as brand of "Gibson Mastermix" used. ie NEBuilder Hi-Fi DNA Assembly Mix will have a different optimal overlap length than NEB Gibson Master Mix and a different optimal length for homebrew Gibson Master Mix (recipe attached at bottom). Generally, 20bp overlap with proper a Tm is suitable.

For example if one was to make a construct that was Seg1-Seg2-Seg3 from individual PCRs of Seg1, Seg2, and Seg3 (as indicated in the figure below) the 3' end of Seg1 would need at least 20bp of homology to the 5' end of Seg2 and Seg3 would require its 5' end to have at least 20bp of homology to Seg2. For constructing a plasmid, use a linearized vector backbone as one of your segments.

Gibson_Assembly_pic.jpg

 

Supplies

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If master mix aliquots are available, and less than 1 year old:
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If homemade master mix aliquots are available, and less than 1 year old:
 
  • 15uL aliquots or a 2X stock of Gibson master mix. Tubes are in both a 96 well holder, and a 50mL tube.
  • Pure water
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Primer Design Using Gibson

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For a thorough discussion on the construction of primers for use in Gibson Assembly, please see the following publication: http://www.ncbi.nlm.nih.gov/pubmed/21601685. GibsonPrimerDesign.jpg
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For a thorough discussion on the construction of primers for use in Gibson Assembly, please see the following publication: http://www.ncbi.nlm.nih.gov/pubmed/21601685. GibsonPrimerDesign.jpg
 

Protocol

  1. Use PCR to produce the DNA segments needed for assembling the new construct. Confirm the success of each PCR by running 5uL of the reaction on an agarose gel.
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  1. Mix 10ng-100ng of each of your DNA segments together (such that their ratios are equimolar) into a 5uL total volume. Therefore, the length of each fragment, and the concentration of the fragments must be taken into account.
  2. Add that 5uL of DNA to the Gibson Master Mix, mix well by pipetting.
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  1. Mix 10ng-100ng of each of your DNA segments together (such that their ratios are equimolar) into a 5uL total volume. Therefore, the length of each fragment, and the concentration of the fragments must be taken into account. Attached at the bottom of this page is an excel spread sheet calculator for an easy and accurate calculation of amount of DNA needed.
  2. Add that 5uL of DNA to 5 uL of Gibson Master Mix, mix well by pipetting (different volumes may be used, but this amount allows for accurate pipetting and reduces the total amount of Gibson Master mix used)
 
  1. Incubate the reaction at 50C for 1 hour.
  2. Dilute the reaction 1:5 in sterile, nuclease free water. Use this diluted sample for PCR (if linear) or transformation (if a plasmid).
  3. Run an aliquot of your final reaction on a gel to verify the presence of your construct. It may be used to do make a 'no incubation' control to determine if the chemistry of the reaction happened.
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 Using 5uL of this reaction provides approximately 1ng/100bp of each fragment in the Gibson Assembly reaction.

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