Revision as of 08:38, 25 July 2011

Gibson assembly

Back to protocols.

Take the different DNA parts you want to assemble and measure their concentration with Nanodrop
We need equal molecuar ratios! Divide the concentration measured above [ng/ul] by the length of each part to have equivalent numbers
Based on these numbers, calculate the ul you need of each to have the same amount of molecules
Take 5 ul of the mixed solution and add 15 ul of Gibson mastermix
Heat at 55°C for 45 minutes (there is a programm written in the iGEM folder)
Purify with Qiagen PCR purification kit

Now the samples are ready to be transformed.

PS: if someone has a less complicated method, please change the protocol :)

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 {{:Team:EPF-Lausanne/Templates/ProtocolHeader|title=Gibson assembly}}
-[[File:EPFL2011 GibsonAssemblystrandscoloured.png]]
+[[File:EPFL2011 GibsonAssemblystrandscoloured.png|700px]]
 Protocol adapted from Nikolaus Obholzer (https://wiki.med.harvard.edu/SysBio/Megason/IsothermalAssembly)