(r8) ProtocolsGibsonCloning < Lab

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---+Gibson Assembly-Work In Progress, do not use yet

---++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

If mastermix aliquots are available, and less than 1 year old:

   * 15uL aliquots of Gibson master mix, located on the top shelf of the -20°C in Welch. Tubes are in both a 96 well holder, and a 50mL tube.
   * Pure water
   * Positive control: 2 overlapping halves of pUC19 (one has AmpR, the other ori).  ATH pUC19 1841-1865 "R", ATH pUC19 306-289 "F"

If no mastermix aliquots remain, but there is still <1 year old isothermal (ISO) reaction buffer available:




If no mastermix aliquots remain, and there is no ISO buffer, or the current ISO buffer is more than a year old:



---++Primer Design Using Gibthon



---++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.
   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.
   1 Add that 5uL of DNA to the Gibson Master Mix, mix well by pipetting. 
   1 Incubate the reaction at 50C for 1 hour.
   1 Dilute the reaction 1:5 in sterile, nuclease free water. Use this diluted sample for PCR (if linear) or transformation (if a plasmid).
   1 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

The positive control should yield ampicillin resistant colonies when transformed into E. coli.

---++ Positive control details

PCR using these primer pairs and pUC19 as the DNA template:

AmpR half of pUC19, ~1150bp
   * ATH pUC19 1841-1865 "R", 5’-CATGACCAAAATCCCTTAACGTGAG-3’
   * ATH pUC19 306-289 "F" 5’-GTAAAACGACGGCCAGTG-3’

Ori half of pUC19, ~1600bp
   * ATH pUC19 1883-1861 "F" 5’-CGCTCAGTGGAACGAAAACTCAC-3’
   * ATH pUC19 266-283 "R" 5’-TCCCCGGGTACCGAGCTC-3’

The following PCR program works if using phusion polymerase, and will work for synthesizing both halves.

Initial denaturation: 98°C for 30 seconds
25 Cycles of: 98°C for 10s, 57°C for 20s, 72°C for 60s
final extension: 72°C for 5 minutes




-- Main.NeilGottel - 13 Feb 2013
Topic revision: r8 - 2013-03-07 - NeilGottel