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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

Access the old non-kit golden gate assembly protocols here

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 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

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-- Main.KateElston - 29 Jan 2018
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Contributors to this topic Edit topic KateElston, PatrickLariviere, SeanLeonard, VictorLi
Topic revision: r7 - 2021-11-03 - 19:56:23 - Main.VictorLi
 
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