Golden Gate cloning

Design checklist

• Insert's restriction sites located within the primer biding region and sufficiently away from the ends of a fragment: Cleavage Close to the End of DNA Fragments | NEB
• Dropout's restriction sites located within the dropout region
• Non-palindromic overhangs
• Ideally overhangs should be completely different such that no amount of hybridization can occur.
• Dropout region cannot have recognition sites too close to each other because otherwise two restriction enzymes might not be able to fit next to each other and cut simultaneously. Keep at least 10 nt between the recognition sites.
• Restriction enzymes from a list of enzymes cited for use in Golden Gate Assembly: see stared enzymes in Type IIS Restriction Enzymes | NEB
• For verification:
  • A unique restriction site in the insert (if possible)
  • A unique restriction site in the dropout
  • A unique restriction site in the backbone
• None of the restriction enzymes should be sensitive to methylation:
  • NEBuffer Activity/Performance Chart with Restriction Enzymes | NEB
  • Effects of CpG Methylation on Restriction Enzyme Cleavage | NEB
  • Dam and Dcm Methylases of E. coli | NEB
• Verify using NEBridge Golden Gate

Procedure

1. Set up a 10 uL reaction:

Reagent	Stock conc.	Range of final amount	Common final amount	Common vol. (uL)	Notes
T4 DNA Ligase Buffer	10X	1X	1X	1	Contains DTT, vortex thoroughly
Backbone		1-4 nM	1.25 nM		Total DNA <1/2 reaction volume; 2:1 ratio of insert:backbone (could go up to 5:1)
Insert		2X backbone	2.5 nM		PCR-amplified and purified (purification can be skipped if cloning a single insert)
T4 DNA Ligase	400 U/uL	20-100 U/uL	40 U/uL	1	More ligase => more misligation; best to avoid high concentration stock ligase
Type IIS restriction enzyme	20 U/uL	.2-1 U/uL	1 U/uL	.5	Both enzymes <10% reaction volume
MilliQ		to 10-20 uL		to 10

• Control conditions (optional):

  • No insert – for checking the background of false positives
  • Backbone + water only – for checking if the transformation worked at all

• Incubate in a thermocycler (lid: 75C):

  • Single insert: 37C - 5 min
  • Library: 37C - 1 hour
  • Multiple inserts or non-BsaI:
    • Initial digestion: 37C - 5 min
    • Cycle 30 times (can go up to 60 for diminishing returns):
      • Annealing: 16C - 1-5 min (choose 5 min to be safe)
      • Digestion: 37C - 1-5 min (choose 5 min to be safe) – make sure to finish with digestion to avoid backbone re-ligation
• Incubate at 65-80C (depends on restriction enzyme) for 20 min to inactivate T4 ligase and restriction enzyme.
• (Optional) Add fresh restriction enzyme, incubate at 37C for 5 min and heat inactivate at 65-80C for 10 min.
  • Helps to digest any empty backbone that may still remain.
• Chill for 5 min to 4C for electroporation or chemical transformation.
• Verification options:
  1. After ligation: Run agarose gel electrophoresis, comparing the backbone and the ligation product. The ligation product is expected to show more bands Make sure to use an SDS-containing loading dye, not TriTrack.
  2. After transformation: Perform colony PCR. Either insert or dropout region must have unique primer sites. After PCR, run index to visualize which colonies had the expected sites.
  3. After overnight culture + plasmid purification:
     1. Perform a restriction analysis:
        • Backbone vs plasmid after GG: determines if the size is correct
        • Linearized backbone vs linearized plasmid after GG using backbone restriction site vs linearized plasmid after GG using GG restriction site vs linearized plasmid after GG using dropout restriction site: determines if the insert is present and if there is only
        • Linearized backbone vs linearized plasmid after GG using GG restriction site
     2. Send off for sequencing

Resources

• Golden Gate Assembly Kit | NEB
• Golden Gate (24 Fragment) Assembly Protocol | NEB
• 13-Fragment Golden Gate Assembly Protocol using SapI | NEB
• Golden Gate Assembly | Bennett Lab Wiki