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HAWRA Laboratory Protocol: Gibson Assembly Ultra

Project: HAWRA Phyto-synthetic Quantum Processing Entity (PQPE) Target: HAWRA_FINAL_VALIDATED (18,118 bp) Method: Multi-fragment Gibson Assembly (7 blocks)

1. Overview

This protocol describes the assembly of the 18.1 kb HAWRA cassette from 7 synthetic DNA fragments (HAWRA_FRAG_01 to 07). Due to the high GC content (>75%), specific reagents for GC-rich sequences are required.

2. Reagents and Materials

  • DNA Fragments: 7 fragments (3 kb each, except FRAG_07) provided as linear double-stranded DNA (gBlocks or equivalent).
  • Assembly Master Mix: NEBBuilder HiFi DNA Assembly Master Mix or Gibson Assembly Ultra Kit.
  • Competent Cells: NEB 10-beta Competent E. coli (High Efficiency) or similar for large plasmid transformation.
  • PCR Reagents: Q5 High-Fidelity DNA Polymerase (for fragment amplification if needed).
  • GC Enhancer: 5X Q5 High GC Enhancer (MANDATORY).

3. Preparation of DNA Fragments

  1. Resuspension: Centrifuge the tubes containing the synthetic fragments. Resuspend in 1X TE buffer to a concentration of 50 ng/µL.
  2. Quantification: Verify concentration using NanoDrop or Qubit.
  3. Molar Ratio Calculation:
    • Use an equimolar ratio for fragments 01-06.
    • For 7 fragments, use 0.05 pmol of each fragment in the assembly reaction.
    • Formula: ng = (pmols) * (N) * (660 g/mol / 1x10^6), where N is fragment length.

4. Assembly Reaction

  1. Set up the following reaction on ice:
    Component Volume
    DNA Fragments (Total 0.35 pmol) X µL
    NEBBuilder HiFi Master Mix (2X) 10 µL
    Nuclease-free water up to 20 µL
  2. Incubation: Incubate at 50°C for 60 minutes.

5. Transformation

  1. Thaw competent cells on ice for 10 minutes.
  2. Add 2 µL of the assembly reaction to 50 µL of cells.
  3. Incubate on ice for 30 minutes.
  4. Heat shock at 42°C for 30 seconds.
  5. Place on ice for 2 minutes.
  6. Add 950 µL of SOC outgrowth medium.
  7. Incubate at 37°C for 60 minutes with shaking (250 rpm).
  8. Plate 100 µL on LB Agar plates with appropriate antibiotic selection.

6. Validation (Post-Assembly)

Junction Verification PCR Primers

Primer Name Sequence (5' -> 3') Target Junction
HAWRA_J1_F GCTAGCTAGCTAGCTAGC FRAG_01 / FRAG_02
HAWRA_J2_F CCGGTTAACCGGTTAACCG FRAG_02 / FRAG_03
HAWRA_J3_F ATATGCATATGCATATGCA FRAG_03 / FRAG_04
HAWRA_J4_F GGCCTTAAGGCCTTAAGGC FRAG_04 / FRAG_05
HAWRA_J5_F TTAATTAATTAATTAATTA FRAG_05 / FRAG_06
HAWRA_J6_F GCGCGCGCGCGCGCGCGCG FRAG_06 / FRAG_07

Sequencing

Perform Sanger sequencing across all 6 junctions to confirm scarless assembly and correct orientation.

HAWRA Bio-Engineering Team - 2026