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> ...)
(Gibson Method, aka VLIC, aka one pot isothermal assembly)
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=Gibson Method, aka VLIC, aka one pot isothermal assembly=
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==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>
(Shamelessly stolen and modified from [[JCA_OPIA | Chris's Version]]).<br>

Revision as of 21:53, 20 June 2010

Gibson Method, aka VLIC, aka one pot isothermal assembly

Original protocol from: this paper, doi:10.1038/nmeth.1318
(Shamelessly stolen and modified from Chris's Version).

Prepare a master mix, as detailed below. Store them in 15 ul aliquots at -20 °C. Then:

  1. Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
  2. 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).
  3. Incubate at 50 °C for 15 to 60 min (60 min is optimal).
  4. Transform as usual
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