Back to Golden Gate Protocols

Second Stage Assembly

Second Stage assemblies are used to build plasmids out of multiple transcriptional units (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 (Promega)
  • 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 42C 1.5 min
2 16C 3 min
Cycles 1-2: Repeat 25x  
3 50C 5 min
4 80C 10 min

  • Transform 2 μL assembly reaction and plate recovery on LB + Selective Antibiotic

Check out troubleshooting tips here

-- Main.KateElston - 29 Jan 2018

 Barrick Lab  >  BroadHostRangeToolkit  >  ProtocolsBTKAssembleMultipleTUPlasmid

Topic revision: r4 - 22 Mar 2018 - 19:26:14 - Main.KateElston
This site is powered by the TWiki collaboration platformCopyright ©2018 Barrick Lab contributing authors. Ideas, requests, problems? Send feedback