(r4) ProtocolsBTKAssembleMultipleTUPlasmid < Lab

[[http://barricklab.org/twiki/bin/view/Lab/GoldenGateAssemblyProtocolsMainPage Back to Golden Gate Protocols]]

---+ 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 &mu;L; 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 &mu;L of 10× T4 DNA ligase buffer (Promega)
   * 1 &mu;L of BsmBI
   * 1 &mu;L of T4 DNA ligase
   * x &mu;L water up to 20 &mu;L total.
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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 &mu;L assembly reaction and plate recovery on LB + Selective Antibiotic

[[http://barricklab.org/twiki/bin/view/Lab/ProtocolsGoldenGateTroubleshooting Check out troubleshooting tips here]]


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-- Main.KateElston - 29 Jan 2018
Barrick Lab > BroadHostRangeToolkit > ProtocolsBTKAssembleMultipleTUPlasmid
Topic revision: r4 - 2018-03-22 - 19:26:14 - Main.KateElston