Gibson Assembly: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(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> ...) |
(No difference)
|
Revision as of 18:52, 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:
- 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).
- Incubate at 50 °C for 15 to 60 min (60 min is optimal).
- Transform as usual
