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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.

Protocol source: NEB (https://www.neb.com/protocols/2020/01/15/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-kit-bsmbi-v2-neb-e1602)

Assembly reaction

Access the old non-kit golden gate assembly protocols here

Total volume will be 20 μL; you will need 50 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.

  • 50 fmol of each transcriptional unit/backbone = 650 * insert length * 50x10^-6 = X ng
  • 2 μL of NEB 10 T4 DNA ligase buffer
  • 1 μL of NEB Golden Gate Enzyme Mix BsmBI-v2
  • x μL water up to 20 μL total.

For easy part volume calculation: GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx


Mix samples well by pipetting, then run the reaction on the thermocycler under the following conditions:

Step Temperature Time
1 42C 1 min
2 16C 1 min
Cycles 1-2: Repeat 30x  
3 60C 5 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: r9 - 2021-11-03 - 20:58:18 - Main.KateElston
 
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