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

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.

• 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

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

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

• x μL water up to 20 μL total.

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

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.

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

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

• 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.

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

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.

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

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

• 1 μL of BsmBI
• 1 μL of T4 DNA ligase
• x μL water up to 20 μL total.

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

Check out troubleshooting tips here

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
• 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:

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

Check out troubleshooting tips here