Team:Edinburgh/GenBrick/GenablerAppendix

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.

Pre-assembly

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

Assembly

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)