Sanger and Nanopore Sequence Verification of Assembled Genetic Constructs Following Golden Gate Cloning in E. coli

For each assembly, pick 8 colonies. All 8 undergo colony PCR; 4 PCR-positive clones proceed to miniprep. Each miniprep is Sanger-sequenced across every designed junction (forward + reverse primers) and one clone is whole-plasmid Nanopore sequenced. Include a no-template PCR control and a known-good reference plasmid control on each run. Replicate the full workflow across 3 independent assemblies to estimate correct-clone rate.

BSL-1 (DH5-alpha). PPE: lab coat, gloves, goggles. SYBR-safe and ethidium-free staining preferred; if any intercalating dye used, treat as a mutagen and dispose appropriately. UV transilluminator requires face/eye shielding. Antibiotics are irritants. Autoclave all culture and plate waste; decontaminate spills with 10% bleach. Nanopore flow cells contain proprietary buffers — dispose per kit SDS.

Positive control: a previously sequence-verified reference plasmid carried through PCR and sequencing confirms primers, reagents, and pipeline. Negative control: no-template (water) colony PCR detects contamination/primer-dimer. Backbone-only/empty-vector control distinguishes uncut backbone carryover from correct assembly. Barcode/no-insert Nanopore control verifies basecalling and demultiplexing. In silico reference defines the expected sequence for alignment.

Correct-clone rate typically 60-95% for well-designed Golden Gate. Colony-PCR positives show a single band of expected size. Sanger reads (Phred Q20 window) match all junction scars with no frameshifts. Nanopore whole-plasmid consensus achieves >50x coverage and >=Q40 consensus, identity >99.9% to reference, with no structural misassembly. Off-target BsaI sites absent.

• E. coli DH5-alpha competent cells
• Golden Gate assembly product (BsaI-HF v2, T4 DNA ligase, designed parts in pYTK/MoClo-style backbones)
• LB agar + chloramphenicol/kanamycin (per backbone)
• Colony PCR master mix (Taq-based), backbone-flanking primers
• Plasmid miniprep kit (silica column)
• Sanger sequencing primers tiling each junction (~500-700 bp spacing)
• Nanopore library prep (Rapid Barcoding Kit) + flow cell (Flongle/MinION)
• Agarose 1%, TAE, DNA ladder, SYBR-safe stain
• Qubit dsDNA HS assay reagents

To confirm correct part assembly and full sequence fidelity of Golden Gate (BsaI Type IIS) constructs transformed into E. coli DH5-alpha, by (1) colony PCR screening, (2) Sanger sequencing across all assembly junctions, and (3) whole-plasmid Oxford Nanopore sequencing to detect indels, point mutations, and misassemblies that junction-only sequencing misses.

1. Transform 2 uL Golden Gate reaction into 50 uL DH5-alpha; recover 1 h at 37 C; plate on selective LB agar; incubate overnight at 37 C.
2. Pick 8 well-isolated colonies; resuspend each in 20 uL water; use 1 uL as colony-PCR template with flanking primers (initial 95 C 5 min denature to lyse).
3. Run PCR products on 1% agarose; identify clones with the expected amplicon size.
4. Inoculate 4 PCR-positive clones into 5 mL selective LB; grow overnight; miniprep and quantify by Qubit.
5. Set up Sanger reactions across each junction (F and R primers); submit for sequencing.
6. Align Sanger reads to the in silico reference; confirm fusion-site scars and ORF reading frames.
7. For 1 verified clone, prepare a Nanopore Rapid Barcoding library (~400 ng plasmid), load on a Flongle/MinION.
8. Basecall (Dorado), assemble/polish whole-plasmid consensus; align to reference.
9. Flag any indel, SNV, or coverage gap; re-pick a clone if misassembled.

Independent: clone identity (colony number) and sequencing method (Sanger vs Nanopore). Dependent: correct-assembly call (junction match, full-length identity, mutation count). Controlled: competent-cell lot, selection antibiotic, PCR program, primer set, DNA input mass, basecaller/version, reference sequence.

Correctly assembled constructs will show expected colony-PCR amplicon sizes, junction Sanger reads matching designed fusion-site scars, and whole-plasmid Nanopore consensus with >=Q40 agreement to the reference, with no off-target BsaI scar reuse or backbone recombination. Misassemblies will be detectable as junction mismatches or coverage anomalies.

Analyze gels by amplicon size vs ladder. Align Sanger AB1 traces to reference in Benchling/SnapGene/ApE; inspect chromatogram quality at junctions. For Nanopore: basecall with Dorado, demultiplex, assemble (Flye or reference-guided), polish (Medaka), align (minimap2), and call variants; visualize coverage in IGV. Report mutation table per clone and final verified status.

1. No colonies: insufficient BsaI digestion or dead competent cells — re-run assembly with fresh enzyme and verify transformation efficiency with a control plasmid. 2. All clones wrong size: primer mismatch or part swap — re-check primer binding sites and part orientation in silico. 3. Sanger junction unreadable: primer too close to junction or secondary structure — design primers 50-100 bp upstream and add DMSO. 4. Low Nanopore coverage: insufficient input DNA or degraded flow cell — increase input to 400 ng and check pore count. 5. Mixed Sanger trace: colony not clonal — re-streak and re-pick a single colony.

Estimate correct-clone proportion with a binomial (Wilson) 95% CI across the 3 assemblies (n=12 clones). Compare Sanger vs Nanopore mutation detection by McNemar's test on discordant calls; alpha=0.05. Power: to detect a clone-quality difference between assembly designs of 30 percentage points at 80% power requires ~36 clones; otherwise report descriptive proportions with CIs. Consensus accuracy reported as Phred Q.