Gibson Cloning-Work In Progress (Neil)

Background and Design

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 it 5' end to have 40bp of homology to Seg2. For constructing a plasmid, use a linearized vector backbone as one of your segments.

Supplies

• Gibson Master Mix from NEB

Protocol

This protocol assumes the presence of Gibson Master Mix.

1. Ensure the water bath is at 50C.
2. Obtain one 15uL aliquot of Gibson Master Mix, keep it on ice,
3. Mix 10ng-100ng of each of your DNA segments together (such that their ratios are equimolar) into a 5uL total volume.
4. Add that 5uL of DNA to the Gibson Master Mix, mix well by pipetting.
5. Incubate the reaction at 50C for 1 hour.
6. Dilute the reaction 1:5 in sterile, nuclease free water. Use this diluted sample for PCR (if linear) or transformation (if a plasmid).
7. 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.

Expected Results

Common Problems/Troubleshooting

-- Main.BrianRenda - 26 Sep 2011 -- Main.NeilGottel - 10 Jan 2013