Genbrick Assembly

Design and Preparation - Linker and Segment

1. Overview
  • Utilise the linker designer software from the EdiGEM 2013 wiki to design Eye and Hook linker oligo pairs
  • Linker and Segment oligos can be custom-made as single-stranded, unphosphorylated DNA
  • Forward and reverse oligo pairs are mixed and phosphorylated prior to annealing

2. Preparation


  • Re-suspend oligo in nuclease-free water to 100 µM (as per instructions)
  • Phosphorylation reaction (not required if oligos with 5' phosphate are ordered)
  • Annealing is achieved by addition of 5 µL 5 M NaCl (50 mM [final]) prior to heat denaturation at < 95°C and slow cooling to Room temperature.



1. Eye-Part-Hook Preparation
a) Using 3-part pathway as an example:
  • Acceptor Vector cassette (+promoter); Acc_RFP
  • LacZ truncated gene (+PLac);PLac_LacZ
  • Green fluorescent protein; GFP
b) Digestion-Ligation reaction of Eye-Part-Hook (E-P-H) (Overnight)
  • Acc_RFP-E + Acc_RFP-P + GFP-H
  • PLac_LacZ-E + PLac_LacZ-P + Acc_RFP-H
  • GFP-E + GFP-P + PLac_LacZ-H


b) c) Purification of Digestion-Ligation E-P-H product to remove non-ligated DNA/plasmid
  • Run 50 µL E-P-H Digestion-Ligation reaction on 1% agarose gel
  • Or: QIAquick PCR Purification kit can be used if Part plasmid does not carry resistance/marker


1. Incubation
  • Use 5 µL each E-P-H required for assembly
  • Add x µl 10X NEBuffer 4 to make 1X final concentration
  • Incubate 30-60 minutes at Room Temperature
  • 3-part Example:
5 µL Acc_RFP E-P-H ([Acc_RFP -E]+[ Acc_RFP -P]+[GFP-H])
5 µL PLac_LacZ E-P-H ([PLac_LacZ-E]+[PLac_LacZ-P]+[Acc_RFP-H)
5 µL GFP E-P-H ([GFP-E]+[GFP-P]+[PLac_LacZ-H])
2 µl NEBuffer 4
3 µL sterile water
  • If larger number of E-P-H in assembly, adjust 10X NEBuffer 4 and water accordingly
2. Transformation
  • Use 10 µL assembly mix to transform 50 µL NEB 10-beta competent cells C3019H
  • Culture above assembly example on LB agar plates with chloramphenicol and IPTG
  • Incubate overnight at 37°C (further growth at RT if colonies require)