Sanger: pUC19-GFP-A206K verification (fwd)

{
    "experiment": {
        "id": 314,
        "custom_id": "SNG-pUC19-A206K-062",
        "title": "Sanger: pUC19-GFP-A206K verification (fwd)",
        "template": "Sanger Sequencing",
        "template_version": "v3.2",
        "category": "Genetics & Genomics",
        "status": "Complete",
        "started_on": "2026-07-21",
        "tags": [
            "sanger-sequencing",
            "AB1-trace",
            "mutation-verification",
            "BigDye",
            "pUC19-GFP"
        ]
    },
    "main_text": "This template captures Sanger sequencing verification of a site-directed A206K monomerizing mutation introduced into the pUC19-GFP construct, confirming the intended single-codon change before downstream use in monomeric GFP applications.\nGoal: verify that the QuikChange mutagenesis introduced only the intended C→G substitution at position 226 (encoding Ala206 → Lys), with no secondary mutations across the 700 bp Sanger read window covering the GFP open reading frame.\nMethod overview: 200 ng/µL miniprep DNA of clone 4 (from pUC19-GFP-A206K mutagenesis EXP-2026-0529) is mixed with 1 µL of 5 µM pUC19-fwd primer (5′-GTAAAACGACGGCCAGT-3′) in 12 µL total volume, submitted to Eurofins LIGHTrun via overnight courier. Sanger reaction uses BigDye Terminator v3.1 chemistry on an ABI 3730xl sequencer with 50 cm capillary array. AB1 trace file downloaded next morning, opened in SnapGene, base-called quality assessed (Q30 above 95% expected), 5′ and 3′ ends trimmed for low-quality regions, and the read aligned against the in silico-predicted pUC19-GFP-A206K reference sequence.\nExpected: 720-880 bp usable read length post-trim; Q30 above 95%; clean single peaks (no overlapping secondary peaks indicating mixed template); intended C→G mutation observed at position 226; no other mismatches across the 700 bp window; alignment confirmation that the construct matches the intended sequence end-to-end.",
    "extra_fields": {
        "Submission": {
            "Template": {
                "type": "select",
                "value": "plasmid miniprep, PCR product, genomic DNA"
            },
            "Primer": {
                "type": "text",
                "value": "pUC19-fwd (M13/pUC)"
            },
            "Service provider": {
                "type": "select",
                "value": "Eurofins, GENEWIZ/Azenta, Macrogen, in-house ABI 3730"
            },
            "Chemistry": {
                "type": "select",
                "value": "BigDye v3.1, BigDye v3.0, BigDye v1.1"
            }
        },
        "Quality": {
            "Total read length": {
                "type": "number",
                "value": "892 bp"
            },
            "Usable read length": {
                "type": "number",
                "value": "852 bp (post-trim)"
            },
            "Q30": {
                "type": "number",
                "value": "98 %"
            },
            "Trim (5'/3')": {
                "type": "text",
                "value": "16 / 24 bp"
            }
        },
        "Mutation Calling": {
            "Intended mutation": {
                "type": "text",
                "value": "C→G @ position 226 (Ala206→Lys)"
            },
            "Observed mutation": {
                "type": "text",
                "value": "C→G @ position 226 confirmed"
            },
            "Other mismatches": {
                "type": "text",
                "value": "none"
            },
            "Call": {
                "type": "select",
                "value": "PASS, FAIL, RETRY"
            }
        }
    },
    "attached_files": [
        {
            "filename": "clone4_fwd.ab1",
            "type": "docx",
            "size": "184 KB"
        },
        {
            "filename": "alignment_snapgene.pdf",
            "type": "pdf",
            "size": "422 KB"
        },
        {
            "filename": "submission_form.pdf",
            "type": "pdf",
            "size": "88 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Confirm miniprep concentration of clone 4: 218 ng/µL, A₂₆₀/A₂₈₀ 1.86",
            "duration": "0:05",
            "complete": true,
            "note": "Within spec for Sanger"
        },
        {
            "n": 2,
            "description": "Select primer: pUC19-fwd (5'-GTAAAACGACGGCCAGT-3') based on insert location 5' of GFP CDS",
            "duration": "0:03",
            "complete": true,
            "note": "Lit Lit-018 binding site"
        },
        {
            "n": 3,
            "description": "Dilute primer to 5 µM working stock in nuclease-free water",
            "duration": "0:03",
            "complete": true
        },
        {
            "n": 4,
            "description": "Mix submission: 5 µL plasmid (200 ng/µL) + 1 µL primer (5 µM) + 6 µL H₂O = 12 µL",
            "duration": "0:05",
            "complete": true
        },
        {
            "n": 5,
            "description": "Label tube with Eurofins barcode SNG-2026-0604-A14, place in submission tube rack",
            "duration": "0:03",
            "complete": true
        },
        {
            "n": 6,
            "description": "Submit via Eurofins LIGHTrun overnight (drop-off by 17:00)",
            "duration": "0:05",
            "complete": true,
            "note": "Cutoff 17:00 met"
        },
        {
            "n": 7,
            "description": "Receive AB1 file next morning via Eurofins portal email + download to project folder",
            "duration": "0:02",
            "complete": true,
            "note": "Received 2026-06-05 09:14"
        },
        {
            "n": 8,
            "description": "Open AB1 in SnapGene; visually inspect chromatogram for peak quality",
            "duration": "0:08",
            "complete": true,
            "note": "Clean single peaks throughout"
        },
        {
            "n": 9,
            "description": "Trim 5' (16 bp) and 3' (24 bp) for low-quality, align to in silico reference",
            "duration": "0:10",
            "complete": true,
            "note": "852 bp usable · Q30 98%"
        },
        {
            "n": 10,
            "description": "Confirm intended C→G mutation at position 226; check no other mismatches; archive AB1 + alignment",
            "duration": "0:08",
            "complete": true,
            "note": "PASS · clone 4 confirmed A206K"
        }
    ],
    "spreadsheet": [
        [
            "Submission",
            "Clone",
            "Primer",
            "Read len",
            "Q30",
            "Intended",
            "Observed",
            "Call"
        ],
        [
            "SNG-A14",
            "clone 1",
            "pUC19-fwd",
            "845",
            "96%",
            "C→G @ pos 226",
            "no edit (WT)",
            "FAIL"
        ],
        [
            "SNG-A15",
            "clone 2",
            "pUC19-fwd",
            "871",
            "98%",
            "C→G @ pos 226",
            "no edit (WT)",
            "FAIL"
        ],
        [
            "SNG-A16",
            "clone 3",
            "pUC19-fwd",
            "812",
            "94%",
            "C→G @ pos 226",
            "C→G + frameshift",
            "FAIL"
        ],
        [
            "SNG-A17",
            "clone 4",
            "pUC19-fwd",
            "852",
            "98%",
            "C→G @ pos 226",
            "C→G confirmed",
            "PASS"
        ],
        [
            "SNG-A18",
            "clone 5",
            "pUC19-fwd",
            "864",
            "97%",
            "C→G @ pos 226",
            "C→G confirmed",
            "PASS"
        ],
        [
            "SNG-A19",
            "clone 6",
            "pUC19-fwd",
            "826",
            "96%",
            "C→G @ pos 226",
            "no edit (WT)",
            "FAIL"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0529",
                "title": "QuikChange A206K mutagenesis (parent clone)",
                "owner": "A. Marin",
                "date": "May 29"
            },
            {
                "ref_id": "EXP-2026-0610",
                "title": "Confirmed clone 4 + 5 scale-up midiprep",
                "owner": "A. Marin",
                "date": "Jun 10"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0142",
                "title": "Sanger Sequencing Submission SOP v1.4",
                "owner": "QA Team",
                "date": "Jan 11"
            },
            {
                "ref_id": "PRIMER-018",
                "title": "pUC19-fwd / M13 primer set (DB entry)",
                "owner": "Primer DB",
                "date": "Aug 4"
            },
            {
                "ref_id": "PLM-0048-K",
                "title": "pUC19-GFP-A206K expected sequence + map",
                "owner": "Plasmid DB",
                "date": "May 30"
            }
        ]
    },
    "compounds": [
        {
            "name": "pUC19-GFP-A206K clone 4 miniprep DNA",
            "catalog_lot": "internal · 218 ng/µL · Lot MP-2026-06-018",
            "stock_status": "In stock"
        },
        {
            "name": "pUC19-fwd primer (M13/pUC universal forward)",
            "catalog_lot": "IDT 018a · Lot 20240118",
            "stock_status": "In stock"
        },
        {
            "name": "Nuclease-free water (Ambion)",
            "catalog_lot": "AM9938 · Lot 24084218",
            "stock_status": "In stock"
        },
        {
            "name": "Eurofins LIGHTrun submission tubes",
            "catalog_lot": "LR-001 · sleeve A-2026",
            "stock_status": "Low"
        }
    ],
    "storage": [
        {
            "name": "Plasmid stocks (A206K clones 1-6)",
            "location": "-20 °C · Freezer B-1 · Drawer 3 · Box A206K-2026",
            "count": "×6"
        },
        {
            "name": "AB1 trace archive (all submissions)",
            "location": "Cloud · /sanger/2026-06/A206K/",
            "count": "×6"
        },
        {
            "name": "Primer working stock (5 µM, 10 µL aliquots)",
            "location": "-20 °C · Freezer B-2 · Rack 1 · Box PR-018",
            "count": "×4"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "Marin Lab"
            },
            {
                "type": "Person",
                "name": "A. Marin"
            }
        ],
        "can_write": [
            {
                "type": "Person",
                "name": "A. Marin"
            },
            {
                "type": "Person",
                "name": "Dr. J. Park"
            }
        ]
    }
}