---+ Second Stage Assembly Second Stage assemblies are used to build plasmids out of multiple transcriptional units ([[http://barricklab.org/twiki/bin/view/Lab/ProtocolsBTKAssembleSingleTUPlasmid Protocol found here]]). Occasionally this assembly can be used as a second step to building troublesome first stage plasmids - the first stage plasmid is assembled in a high copy/easier to work with backbone then moved to lower copy/more difficult to work with backbone. This assembly makes use of the BsmBI enzyme as described below. ---+++ Assembly reaction Total volume will be 20 μL; you will need 10 fmol of each transcriptional unit plasmid for this assembly. Various backbones are regularly used in this step - if toxicity issues may be a factor, lower copy number vectors are recommended. * 10 fmol of each transcriptional unit/backbone * 2 μL of 10× T4 DNA ligase buffer * 1 μL of BsmBI * 1 μL of T4 DNA ligase * x μL water up to 20 μL total. Mix samples well by pipetting, then run the reaction on the thermocycler under the following conditions: | *Step* | *Temperature* | *Time* | | 1 | 42°C | 1.5 min | | 2 | 16°C | 3 min | | Cycles 1-2: | Repeat 25x | | | 3 | 50°C | 5 min | | 4 | 80°C | 10 min | * Transform 2 μL assembly reaction and plate recovery on LB + Selective Antibiotic [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsGoldenGateTroubleshooting Check out troubleshooting tips here]] [[http://barricklab.org/twiki/bin/view/Lab/GoldenGateAssemblyProtocolsMainPage Back to Golden Gate Protocols]] ---------------------------------------------------------- -- Main.KateElston - 29 Jan 2018
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Topic revision: r2 - 2018-01-29 - KateElston