First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Protocol source: NEB (https://www.neb.com/protocols/2018/10/02/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1601)

Assembly reaction

Total volume will be 20 μL; you will need 50 fmol of each part plasmid for this assembly. Typically the pYTK095 Type 6-8 plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 2 μL of NEB 10× T4 DNA ligase buffer
• 1 μL of NEB Golden Gate Enzyme Mix BsaI-HFv2 (*DO NOT use BsaI or BsaI-HF)
• x μL water up to 20 μL total.

For easy part volume calculation: GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx

*BsaI and BsaI-HF don't have high fidelity and/or activity under the Golden Gate buffer/temperature conditions.

Transform 2 μL of the assembly reaction and plate on an appropriate antibiotic (Amp/Carb if using pYTK095).

Tips from New England Biolabs on ways to change the reaction conditions for difficult assemblies involving many parts:

• Different reaction times based on the number of inserts (link)
• Better fidelity for assembling many inserts using a constant reaction temperature: (link)

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

• x μL water up to 20 μL total.

For easy part volume calculation: GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx

*BsaI and BsaI-HF don't have high fidelity and/or activity under the Golden Gate buffer/temperature conditions.

Tips from New England Biolabs on ways to change the reaction conditions for difficult assemblies involving many parts:

• Different reaction times based on the number of inserts (link)
• Better fidelity for assembling many inserts using a constant reaction temperature: (link)

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

• 10 fmol of each part plasmid = 650 * insert length * 10x10^-6 = X ng
• 2 μL of 10× T4 DNA ligase buffer (Promega)
• 1 μL of BsaI-HFv2 (*DO NOT use BsaI or BsaI-HF)
• 1 μL of T4 DNA ligase (Promega)
• x μL water up to 20 μL total.

** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency.

Transform 2 μL of the assembly reaction and plate on an appropriate antibiotic (Amp/Carb if using pYTK095).

Tips from New England Biolabs on ways to change the reaction conditions for difficult assemblies involving many parts:

• Different reaction times based on the number of inserts (link)
• Better fidelity for assembling many inserts using a constant reaction temperature: (link)

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pYTK095 Type 6-8 plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid = 650 * insert length * 10x10^-6 = X ng
• 2 μL of 10× T4 DNA ligase buffer (Promega)
• 1 μL of BsaI-HFv2 (*DO NOT use BsaI or BsaI-HF)
• 1 μL of T4 DNA ligase (Promega)
• x μL water up to 20 μL total.

For easy part volume calculation: BroadHostRange_Reaction_Calculator.xlsx

*BsaI and BsaI-HF don't have high fidelity and/or activity under the Golden Gate buffer/temperature conditions.

** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency.

Tips from New England Biolabs on ways to change the reaction conditions for difficult assemblies involving many parts:

• Different reaction times based on the number of inserts (link)
• Better fidelity for assembling many inserts using a constant reaction temperature: (link)

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pYTK095 Type 6-8 plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 2 μL of 10× T4 DNA ligase buffer (Promega)
• 1 μL of BsaI-HFv2 (*DO NOT use BsaI or BsaI-HF)
• 1 μL of T4 DNA ligase (Promega)
• x μL water up to 20 μL total.

** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency.

Transform 2 μL of the assembly reaction and plate on an appropriate antibiotic (Amp/Carb if using pYTK095).

Tips from New England Biolabs on ways to change the reaction conditions for difficult assemblies involving many parts:

• Different reaction times based on the number of inserts (link)
• Better fidelity for assembling many inserts using a constant reaction temperature: (link)

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pYTK095 Type 6-8 plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer (Promega)
• 1 μL of BsaI-HFv2 (*DO NOT use BsaI or BsaI-HF)
• 1 μL of T4 DNA ligase (Promega)
• x μL water up to 20 μL total.

*BsaI and BsaI-HF don't have high fidelity and/or activity under the Golden Gate buffer/temperature conditions.

** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency.

Transform 2 μL of the assembly reaction and plate on an appropriate antibiotic (Amp/Carb if using pYTK095).

Tips from New England Biolabs on ways to change the reaction conditions for difficult assemblies involving many parts:

• Different reaction times based on the number of inserts (link)
• Better fidelity for assembling many inserts using a constant reaction temperature: (link)

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer (Promega)

• 1 μL of T4 DNA ligase (Promega)
• x μL water up to 20 μL total.

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pYTK095 part plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer (Promega)
• 1 μL of BsaI-HFv2 ( DO NOT use BsaI-HF*)
• 1 μL of T4 DNA ligase (Promega)
• x μL water up to 20 μL total.

*BsaI-HF is optimized for different buffer conditions and doesn't work well

** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency.

• Transform 2 μL assembly reaction and plate on appropriate antibiotic

Due to the nature of attempting to assemble a high number of plasmids in one reaction there are often issues that arise: Check out troubleshooting tips here

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer (Promega)
• 1 μL of BsaI-HFv2 ( DO NOT use BsaI-HF*)
• 1 μL of T4 DNA ligase (Promega)
• x μL water up to 20 μL total.

*BsaI-HF is optimized for different buffer conditions and doesn't work well

** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency.

• Transform 2 μL assembly reaction and plate on appropriate antibiotic

Due to the nature of attempting to assemble a high number of plasmids in one reaction there are often issues that arise: Check out troubleshooting tips here

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pBTK095 part plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer (Promega)
• 1 μL of BsaI-HFv2 ( DO NOT use BsaI-HF*)
• 1 μL of T4 DNA ligase (Promega)
• x μL water up to 20 μL total.

*BsaI-HF is optimized for different buffer conditions and doesn't work well

** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency.

• Transform 2 μL assembly reaction and plate on appropriate antibiotic

Due to the nature of attempting to assemble a high number of plasmids in one reaction there are often issues that arise: Check out troubleshooting tips here

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pBTK095 part plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer (Promega)
• 1 μL of BsaI-HFv2 ( DO NOT use BsaI-HF*)

• x μL water up to 20 μL total.

*BsaI-HF is optimized for different buffer conditions and doesn't work well

• Transform 2 μL assembly reaction and plate on appropriate antibiotic

Due to the nature of attempting to assemble a high number of plasmids in one reaction there are often issues that arise: Check out troubleshooting tips here

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pBTK095 part plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid

• 1 μL of BsaI-HFv2 ( DO NOT use BsaI-HF*)
• 1 μL of T4 DNA ligase
• x μL water up to 20 μL total.

• Transform 2 μL assembly reaction and plate on appropriate antibiotic

Due to the nature of attempting to assemble a high number of plasmids in one reaction there are often issues that arise: Check out troubleshooting tips here

First-Stage Plasmid (Transcriptional Unit) Assembly

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pBTK095 part plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer

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

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pBTK095 part plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer
• 1 μL of BsaI (DO NOT use BsaI-HF*)
• 1 μL of T4 DNA ligase
• x μL water up to 20 μL total.

*BsaI-HF has been optimized for different buffer conditions and won't work as well

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

• Transform 2 μL assembly reaction and plate on LB + Carb (alternate backbones may have alternate antibiotic resistances!)

Due to the nature of attempting to assemble a high number of plasmids in one reaction there are often issues that arise: Check out troubleshooting tips here

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pBTK095 part plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer

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

• Transform 2 μL assembly reaction and plate on LB + Carb (alternate backbones may have alternate antibiotic resistances!)

Due to the nature of attempting to assemble a high number of plasmids in one reaction there are often issues that arise: Check out troubleshooting tips here

First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present (Described here).

Assembly reaction

Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pBTK095 part plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

• 10 fmol of each part plasmid
• 2 μL of 10× T4 DNA ligase buffer
• 1 μL of BsaI
• 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 on LB + Carb (alternate backbones may have alternate antibiotic resistances!)

Due to the nature of attempting to assemble a high number of plasmids in one reaction there are often issues that arise: Check out troubleshooting tips here