Two-fragment Gibson assembly of EGFP into pET-28a (NdeI/XhoI site, N-terminal His-tag in frame), from the plan recorded on Gibson plan: EGFP into pET-28a. 25 bp overlaps on each junction.
Estimated time: 2 days (bench time 3 h) · Difficulty: intermediate · Yield: 6,089 bp circular, KanR
Reaction setup: PCR (50 µL, one per fragment)
| Component | Volume | Note |
|---|
| Q5 Hot Start 2× Master Mix | 25 µL | |
| Forward primer, 10 µM | 2.5 µL | bb_F / ins_F from yesterday's design |
| Reverse primer, 10 µM | 2.5 µL | bb_R / ins_R |
| Template | 1 µL | pET-28a 5 ng; EGFP source 5 ng |
| Nuclease-free water | 19 µL | |
Protocol steps
- PCR the two fragments. Duration 2 h · 98 °C / 62 °C / 72 °C 98 °C 30 s; 30 cycles of 98 °C 10 s, 62 °C 20 s, 72 °C 30 s per kb (backbone 5.3 kb: 2 min 40 s; insert 0.7 kb: 25 s); final 72 °C 2 min. Run 5 µL on a 1% gel: expect 5.3 kb and 0.75 kb.
- Treat with DpnI. Duration 1 h · 37 °C Add 1 µL DpnI (20 U) straight into each PCR. 37 °C for 1 h. Removes the methylated template so empty parent vector does not come through.
- Clean up and quantify. Duration 20 min · room temp Column cleanup, elute in 15 µL. Nanodrop both fragments; A260/280 of 1.8 or better.
- Assemble. Duration 60 min · 50 °C Backbone 0.05 pmol (170 ng of 5.3 kb), insert 0.1 pmol (47 ng of 0.72 kb), NEBuilder HiFi 2× Master Mix 10 µL, water to 20 µL. 50 °C for 60 min, then on ice. Include a backbone-only control.
- Transform and plate. Duration 1.5 h + overnight · 42 °C / 37 °C 2 µL of assembly into 50 µL DH5α; ice 30 min, 42 °C 30 s, ice 2 min, 950 µL SOC, 37 °C 1 h shaking. Plate 100 µL on LB + kanamycin 50 µg/mL, 37 °C overnight.
- Screen colonies. Duration 3 h · day 2 Colony PCR on 8 colonies with T7 promoter / T7 terminator primers: expect about 1.0 kb with insert, about 0.3 kb for empty vector. Miniprep two positives and send for Sanger with the T7 primers.
Materials required
| Item | Supplier | Catalog # |
|---|
| NEBuilder HiFi DNA Assembly Master Mix | NEB | E2621 |
| Q5 Hot Start High-Fidelity 2× Master Mix | NEB | M0494 |
| DpnI | NEB | R0176 |
| Monarch PCR & DNA Cleanup Kit | NEB | T1030 |
| DH5α chemically competent E. coli | NEB | C2987 |
| LB agar plates, kanamycin 50 µg/mL | in house | |
Equipment needed
- Thermocycler
- Heat block or water bath at 42 °C and 50 °C
- Shaking incubator, 37 °C
- Spectrophotometer (Nanodrop) and gel electrophoresis setup
Safety notes
- BSL-1 organism; standard microbiological practice, autoclave all cultures and plates before disposal.
- Kanamycin stock is an irritant; weigh in a fume hood and wear gloves.
- Use a non-mutagenic DNA stain; if using ethidium bromide, handle gels with nitrile gloves and dispose as hazardous waste.
Quality control checkpoints
- After PCR. Gel: single band at 5.3 kb (backbone) and 0.75 kb (insert). Pass: no smear, no primer-dimer band.
- After transformation. Colony count: assembly plate at least 10× the backbone-only control. Pass: control under 10 colonies.
- After sequencing. Sanger with T7 primers, aligned to pET-28a-GFP. Pass: 100% identity across the insert and both junctions.
