May 01, 2026
PCR Amplification of Gibson Assembly Fragments for the BM3R1/PhlF Genetic Circuit (V.1)
- Varsha Athreya1,2,
- Melissa Regalado1,2
- 1Boston University;
- 2DAMP Lab
- EC552_Varsha_Melissa
Protocol Citation: Varsha Athreya, Melissa Regalado 2026. PCR Amplification of Gibson Assembly Fragments for the BM3R1/PhlF Genetic Circuit (V.1). protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6j5w5vqe/v1
Manuscript citation:
Forked from: PCR Using Q5^^® High-Fidelity DNA Polymerase (NEB #M0491) — protocols.io
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 01, 2026
Last Modified: May 01, 2026
Protocol Integer ID: 316100
Keywords: PCR, Q5, Gibson Assembly, BM3R1, PhlF, Pxyl, Pxyl-tetO, GFP, pUC19, synthetic biology, EC552, pcr amplification of gibson assembly fragment, second genetic circuit of ec552 hw, fidelity dna polymerase with primer, fidelity dna polymerase, pcr amplification, polymerase chain reaction, second genetic circuit, responsive sensor promoter, gibson assembly, ec552 hw, gibson assembly fragment, bm3r1 x2, puc19 backbone, pcr, dna, dna fragment, linearized puc19 backbone, bp plasmid, gfp reporter
Abstract
This protocol describes the polymerase chain reaction (PCR) amplification of the eight DNA fragments required to construct the second genetic circuit of EC552 HW #2 via Gibson Assembly. The final ~9,210 bp plasmid is built into a linearized pUC19 backbone and contains two xylose-responsive sensor promoters (Pxyl and Pxyl-tetO) feeding two NOT gates (BM3R1 x2 and PhlF) that drive a GFP reporter. Each part (PB1–P2–Pln1–B1–Pln2–B2–PP1–Y) is amplified separately using Q5^^® High-Fidelity DNA Polymerase with primers carrying ≥20 bp Gibson overlaps to its neighboring fragment. Amplicons are verified on a 1% agarose gel, column-purified, and quantified before assembly. This protocol is written to be mapped to the corresponding DAMP Lab operation in the canvas-to-protocols.io workflow.
Guidelines
Q5 High-Fidelity DNA Polymerase has an error rate e100-fold lower than Taq and is recommended for any amplicon that will be cloned. Note that Q5 protocols differ from those of other polymerases — use the conditions below for optimal performance.
- Set up reactions on ice. Q5 (non-hot-start) should be added last.
- Use the NEB Tm Calculator (tmcalculator.neb.com) with the Q5 setting to determine annealing temperature. Typically use 3 °C above the Tm of the lower-Tm primer.
- Extension time: 20–30 s/kb for plasmid templates; 30 s/kb is a safe default for the EC552 fragments (all 2 kb).
- Q5 produces blunt-ended products — compatible with Gibson Assembly directly; no A-tailing needed.
- Q5 cannot incorporate dUTP — do not use uracil-containing primers or templates.
- Always include a no-template control (NTC) per primer pair to detect contamination or primer-dimer artifacts.
Materials
Reagents and equipment required for one full assembly (8 fragments + NTCs):
- Q5 High-Fidelity 2X Master Mix (NEB, M0492S)
- Deoxynucleotide (dNTP) Solution Mix (NEB, N0447S)
- Nuclease-Free Water (NEB / IDT, B1500S)
- Custom oligonucleotide primers (10 µM) (IDT, Custom)
- Quick-Load Purple 1 kb Plus DNA Ladder (NEB, N0550S)
- Monarch Spin PCR DNA Cleanup Kit (NEB, T1130S)
- 0.2 mL thin-walled PCR tubes (sterile)
- Thermocycler (heated lid)
- Microcentrifuge / mini-spin
- Pipettes (P2, P10, P20, P200, P1000)
- Filter pipette tips
- Ice bucket
Safety warnings
- Wear gloves, lab coat, and safety glasses at all times.
- SYBR Safe and ethidium bromide are DNA-intercalating agents — handle gel staining waste per institutional EHS guidelines.
- UV transilluminators emit UV-B/UV-C; use the safety shield and a face shield. Prefer a blue-light transilluminator when available.
- Dispose of biological waste (used tubes, tips, gels) in designated biohazard containers.
Before start
- Thaw 5X Q5 Reaction Buffer, dNTPs, and primer working stocks (10 µM) on ice. Vortex briefly and spin down.
- Keep Q5 polymerase in a benchtop cooler (-20 °C) until immediately before pipetting.
- Pre-cool a PCR tube rack on ice.
- Verify primer Tm values with the NEB Tm Calculator. For each primer pair, record the calculated annealing temperature (Ta) in your run record.
- Label one 0.2 mL PCR tube per fragment + one NTC per primer pair (e.g., "1-PB1", "1-PB1-NTC", ... "8-Y", "8-Y-NTC").
- Pre-program the thermocycler with the conditions in Step 4.
Procedure
Add components in the order listed (water → buffer → dNTPs → primers → template → polymerase). Mix gently by flicking; do not vortex once enzyme has been added. Briefly spin to collect liquid at the bottom of the tube.
Aliquot the master mix into each labeled PCR tube.
Add 2.5 µL of the appropriate 10 µM forward primer and 2.5 µL of the corresponding 10 µM reverse primer (final 0.5 µM each).
Add 1 µL of plasmid template (1 pg–1 ng total). For NTCs, substitute 1 µL nuclease-free water for the template.
Cap tubes, flick to mix, and pulse-spin.
If your thermocycler does not have a heated lid, overlay each reaction with ~25 µL mineral oil to prevent evaporation during cycling. Skip this step on any modern heated-lid instrument.
Transfer tubes to a thermocycler preheated to 98 °C and run the following program:
Initial Denaturation: 98 °C, 30 s, 1 cycle
Denaturation: 98 °C, 10 s, 30 cycles
Annealing: 50–72 °C, 20 s, 30 cycles
Extension: 72 °C, 30 s/kb, 30 cycles
Final Extension: 72 °C, 2 min, 1 cycle
Verify amplification by gel electrophoresis
Cast a 1% agarose gel in 1X TAE with SYBR Safe (or equivalent stain).
Load 5 µL of each PCR reaction alongside 5 µL of Quick-Load Purple 1 kb Plus DNA Ladder (NEB N0550S).
Run at 100 V for ~30 min (until the dye front has migrated ~⅔ down the gel).
Image the gel on a UV or blue-light transilluminator. Confirm a single band at the expected size for each fragment, and that all NTC lanes are empty.
Clean up amplicons
Purify each PCR product using the Monarch Spin PCR DNA Cleanup Kit (NEB T1130) per manufacturer instructions. Elute in 20 µL nuclease-free water or elution buffer.
Quantify each cleaned amplicon by Qubit dsDNA HS or NanoDrop. Record concentration (ng/µL) and A260/A280 in the run record.
Store purified fragments at -20 °C until Gibson Assembly. Use within 1 week for best assembly efficiency.
Expected Outcomes
Each PCR reaction yields a single, sharp band on the gel at the expected size for that fragment.
All NTC lanes are clean (no bands).
Post-cleanup yield: typically 10–50 ng/µL in 20 µL elution (200 ng–1 µg total per fragment), sufficient for multiple Gibson Assembly reactions.
A260/A280 ratios between 1.8 and 2.0 indicate clean, protein-free DNA suitable for downstream assembly.
Troubleshooting
No band: Re-check primer Tm calculation; lower the annealing temperature by 2–3 °C; verify template concentration (1 pg–1 ng plasmid); add 1X Q5 High GC Enhancer for GC-rich amplicons (e.g., Pxyl-tetO region).
Multiple bands / smearing: Raise annealing temperature by 2–3 °C; reduce template by 10×; redesign primers to improve specificity; reduce cycle number to 25.
Band in NTC lane: Discard primer aliquots and remake from frozen stock; use filter tips; UV-decontaminate the PCR setup area.
Failed Gibson downstream: Re-run amplicons on a gel after cleanup to confirm no carryover primer-dimer; ensure ≥20 bp Gibson overlaps in primer design; balance fragment molar ratios in the assembly reaction.
Protocol references
- New England Biolabs. PCR Using Q5^^® High-Fidelity DNA Polymerase (M0491). protocols.io. https://dx.doi.org/10.17504/protocols.io.cjtunmn
- New England Biolabs. Gibson Assembly^^® Protocol (E5510 / E2611). https://www.neb.com/en/protocols/2012/12/11/gibson-assembly-protocol-e5510
- NEB Tm Calculator. https://tmcalculator.neb.com
- DAMP Lab Protocols. Boston University Cross-Disciplinary Integration of Design, Automation, and Modeling for Synthetic Biology.
- EC552 Computational Synthetic Biology for Engineers, HW #2 (Athreya Regalado, 2026).