Part Plasmid Assembly

Once you have designed your part and either amplified with PCR or ordered the desired gBlock (as described here) you can proceed to the assembly step of the part plasmid itself. The example reaction below shows pYTK001 used as the entry vector for the reaction; however, this can be substituted for any other entry vector with requisite BsmBI cut sites. Once built, part plasmids can be assembled into transcriptional units.

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

1. Calculate the mass (in ng) required for 50 fmol of vector and 100 fmol of insert using NEB's NEBioCalculator: https://nebiocalculator.neb.com/#!/dsdnaamt

2. Set up the following reaction mix:

• 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

Expected Results

• (If using pYTK001 as your entry vector) Multiple non-fluorescent colonies
  • Be sure to screen colonies for GFP expression on a blue light transilluminator, pick colonies that do not fluoresce!
• Cells containing properly assembled plasmids (confirmed through PCR and Sanger Sequencing)

If you are having repeated issues check out the Troubleshooting page!