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#314 Molecular Biology Complete

Digest gel: pUC19-GFP / EcoRI + BamHI (6 clones)

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agarose-gel restriction-digest EcoRI-BamHI clone-screening pUC19-GFP

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This template captures a diagnostic 1.2% agarose gel screening 6 candidate pUC19-GFP clones for the expected EcoRI + BamHI double-digest pattern.

Goal: identify clones containing the correct GFP insert orientation and full restriction-site integrity by confirming a two-band pattern of 2686 bp (vector backbone) + 720 bp (GFP insert) on agarose gel.

Method overview: 1.2% agarose dissolved in 1× TAE buffer, 5 µL SYBR Safe per 50 mL gel, poured into a Mini-Sub Cell GT tray with an 8-tooth comb. Each well loaded with 10 µL of completed digest reaction + 2 µL of 6× loading dye. Lane 1: 5 µL of 1 kb Plus DNA Ladder. Lanes 2-7: clones 1-6. Lane 8: undigested pUC19-GFP plasmid (control showing supercoiled + nicked). Run: 100 V for 50 minutes, imaging on UV transilluminator with TIFF save.

Expected: Lanes 2, 3, 5, 6 show clean 2686 bp + 720 bp two-band pattern (4 correct clones). Lane 4 shows partial digestion with a third uncut band. Undigested control shows single ~3.4 kb apparent supercoiled + nicked bands. Ladder reference confirms size assignments.

Last saved: 34 seconds ago

10 fields loaded from Gel Electrophoresis baseline (SOP v2.4)

Gel

Agarose concentration

number

1.2%

Buffer

select

1× TAE, 1× TBE, 0.5× TBE

Stain

select

SYBR Safe, GelRed, EtBr

Ladder

select

1 kb Plus, 100 bp, λ HindIII, 1 kb

Run

Voltage

number

100V

Run time

number

50min

Comb teeth

number

8

Load volume

number

12µL

Scoring

Expected bands

text

2686 bp + 720 bp

Pass criteria

text

both bands at expected size

digest_gel_image.tif

3.2 MB

lane_scoring.csv

12 KB

expected_band_map.pdf

186 KB

01

Weigh 0.6 g agarose, mix with 50 mL 1× TAE in flask

1.2% w/v

0:03

02

Microwave 90 s with periodic swirls until fully dissolved

No visible undissolved particles

0:02

03

Cool to ~60°C, add 5 µL SYBR Safe, swirl gently

0:08

04

Pour into tray with 8-tooth comb, let set 30 min at RT

Gel set firm at 30 min

0:30

05

Submerge gel in 1× TAE, remove comb, ensure buffer covers wells

0:03

06

Mix 10 µL digest + 2 µL 6× loading dye per sample on parafilm

0:05

07

Load lane 1: 5 µL ladder; lanes 2-7: clones 1-6; lane 8: undigested control

0:05

08

Run at 100 V for 50 min until dye front 2/3 down

0:50

09

Transfer to UV imager, capture TIFF + auto-exposure

0:04

10

Score per-clone: 4/6 correct (lanes 2,3,5,6); 1 partial (lane 4); 1 fail (lane 7 wrong size)

Decision: scale up clones 2 + 5 for sequencing

0:10

	A	B	C	D	E	F
1	Lane	Sample	Expected (bp)	Observed (bp)	Score	Downstream
2	1	1 kb Plus ladder	reference	reference	—	—
3	2	clone 1	2686 + 720	2686 + 720	PASS	Scale up for sequencing
4	3	clone 2	2686 + 720	2686 + 720	PASS	Scale up for sequencing
5	4	clone 3	2686 + 720	2686 + 720 + uncut	PARTIAL	Re-digest, recheck
6	5	clone 4	2686 + 720	2686 + 720	PASS	Scale up for sequencing
7	6	clone 5	2686 + 720	2686 + 720	PASS	Scale up for sequencing
8	7	clone 6	2686 + 720	wrong size	FAIL	Discard
9	8	undigested ctrl	single supercoiled	~3.4 kb	—	Confirms plasmid stock identity

EXP-2026-0603 EcoRI + BamHI digest setup (6 clones) A. Marin Jun 3

EXP-2026-0606 Clones 2 + 5 Sanger sequencing setup A. Marin Jun 6

SOP-0204 Gel Electrophoresis SOP v2.4 (TAE / 1.2% agarose) QA Team Feb 4

LAD-0001 1 kb Plus DNA Ladder reference image + sizes NEB Docs Aug 12

PLM-0048 pUC19-GFP expected digest map Plasmid DB Sep 4

Agarose, molecular biology grade

A9539 · Lot SLBR8814V

In stock

50× TAE buffer (concentrate)

1610773 · Lot 64158119

In stock

SYBR Safe DNA gel stain (10,000×)

S33102 · Lot 2422184

Low

1 kb Plus DNA Ladder

N3232 · Lot 10174812

In stock

6× DNA Loading Dye (purple)

B7024 · Lot 10173621

In stock

SYBR Safe stain (10,000× working)

4 °C · Fridge 2 · Shelf 1 · Box DNA-STAIN

×4

DNA ladder aliquots (50 µL working)

-20 °C · Freezer B-2 · Rack 1 · Box LAD-2026

×10

Gel image archive (TIF + raw imager output)

Cloud · /gels/2026-06/041/

×1

Visibility

Marin LabA. Marin

Can write

A. MarinDr. J. Park

Color

Stroke width

Eraser

Use Ctrl + click to add text

{
    "experiment": {
        "id": 314,
        "custom_id": "GEL-2026-0604-041",
        "title": "Digest gel: pUC19-GFP / EcoRI + BamHI (6 clones)",
        "template": "Gel Electrophoresis",
        "template_version": "v3.2",
        "category": "Molecular Biology",
        "status": "Complete",
        "started_on": "2026-07-15",
        "tags": [
            "agarose-gel",
            "restriction-digest",
            "EcoRI-BamHI",
            "clone-screening",
            "pUC19-GFP"
        ]
    },
    "main_text": "This template captures a diagnostic 1.2% agarose gel screening 6 candidate pUC19-GFP clones for the expected EcoRI + BamHI double-digest pattern.\nGoal: identify clones containing the correct GFP insert orientation and full restriction-site integrity by confirming a two-band pattern of 2686 bp (vector backbone) + 720 bp (GFP insert) on agarose gel.\nMethod overview: 1.2% agarose dissolved in 1× TAE buffer, 5 µL SYBR Safe per 50 mL gel, poured into a Mini-Sub Cell GT tray with an 8-tooth comb. Each well loaded with 10 µL of completed digest reaction + 2 µL of 6× loading dye. Lane 1: 5 µL of 1 kb Plus DNA Ladder. Lanes 2-7: clones 1-6. Lane 8: undigested pUC19-GFP plasmid (control showing supercoiled + nicked). Run: 100 V for 50 minutes, imaging on UV transilluminator with TIFF save.\nExpected: Lanes 2, 3, 5, 6 show clean 2686 bp + 720 bp two-band pattern (4 correct clones). Lane 4 shows partial digestion with a third uncut band. Undigested control shows single ~3.4 kb apparent supercoiled + nicked bands. Ladder reference confirms size assignments.",
    "extra_fields": {
        "Gel": {
            "Agarose concentration": {
                "type": "number",
                "value": "1.2 %"
            },
            "Buffer": {
                "type": "select",
                "value": "1× TAE, 1× TBE, 0.5× TBE"
            },
            "Stain": {
                "type": "select",
                "value": "SYBR Safe, GelRed, EtBr"
            },
            "Ladder": {
                "type": "select",
                "value": "1 kb Plus, 100 bp, λ HindIII, 1 kb"
            }
        },
        "Run": {
            "Voltage": {
                "type": "number",
                "value": "100 V"
            },
            "Run time": {
                "type": "number",
                "value": "50 min"
            },
            "Comb teeth": {
                "type": "number",
                "value": "8"
            },
            "Load volume": {
                "type": "number",
                "value": "12 µL"
            }
        },
        "Scoring": {
            "Expected bands": {
                "type": "text",
                "value": "2686 bp + 720 bp"
            },
            "Pass criteria": {
                "type": "text",
                "value": "both bands at expected size"
            }
        }
    },
    "attached_files": [
        {
            "filename": "digest_gel_image.tif",
            "type": "png",
            "size": "3.2 MB"
        },
        {
            "filename": "lane_scoring.csv",
            "type": "csv",
            "size": "12 KB"
        },
        {
            "filename": "expected_band_map.pdf",
            "type": "pdf",
            "size": "186 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Weigh 0.6 g agarose, mix with 50 mL 1× TAE in flask",
            "duration": "0:03",
            "complete": true,
            "note": "1.2% w/v"
        },
        {
            "n": 2,
            "description": "Microwave 90 s with periodic swirls until fully dissolved",
            "duration": "0:02",
            "complete": true,
            "note": "No visible undissolved particles"
        },
        {
            "n": 3,
            "description": "Cool to ~60°C, add 5 µL SYBR Safe, swirl gently",
            "duration": "0:08",
            "complete": true
        },
        {
            "n": 4,
            "description": "Pour into tray with 8-tooth comb, let set 30 min at RT",
            "duration": "0:30",
            "complete": true,
            "note": "Gel set firm at 30 min"
        },
        {
            "n": 5,
            "description": "Submerge gel in 1× TAE, remove comb, ensure buffer covers wells",
            "duration": "0:03",
            "complete": true
        },
        {
            "n": 6,
            "description": "Mix 10 µL digest + 2 µL 6× loading dye per sample on parafilm",
            "duration": "0:05",
            "complete": true
        },
        {
            "n": 7,
            "description": "Load lane 1: 5 µL ladder; lanes 2-7: clones 1-6; lane 8: undigested control",
            "duration": "0:05",
            "complete": true
        },
        {
            "n": 8,
            "description": "Run at 100 V for 50 min until dye front 2/3 down",
            "duration": "0:50",
            "complete": true
        },
        {
            "n": 9,
            "description": "Transfer to UV imager, capture TIFF + auto-exposure",
            "duration": "0:04",
            "complete": true
        },
        {
            "n": 10,
            "description": "Score per-clone: 4/6 correct (lanes 2,3,5,6); 1 partial (lane 4); 1 fail (lane 7 wrong size)",
            "duration": "0:10",
            "complete": true,
            "note": "Decision: scale up clones 2 + 5 for sequencing"
        }
    ],
    "spreadsheet": [
        [
            "Lane",
            "Sample",
            "Expected (bp)",
            "Observed (bp)",
            "Score",
            "Downstream"
        ],
        [
            "1",
            "1 kb Plus ladder",
            "reference",
            "reference",
            "—",
            "—"
        ],
        [
            "2",
            "clone 1",
            "2686 + 720",
            "2686 + 720",
            "PASS",
            "Scale up for sequencing"
        ],
        [
            "3",
            "clone 2",
            "2686 + 720",
            "2686 + 720",
            "PASS",
            "Scale up for sequencing"
        ],
        [
            "4",
            "clone 3",
            "2686 + 720",
            "2686 + 720 + uncut",
            "PARTIAL",
            "Re-digest, recheck"
        ],
        [
            "5",
            "clone 4",
            "2686 + 720",
            "2686 + 720",
            "PASS",
            "Scale up for sequencing"
        ],
        [
            "6",
            "clone 5",
            "2686 + 720",
            "2686 + 720",
            "PASS",
            "Scale up for sequencing"
        ],
        [
            "7",
            "clone 6",
            "2686 + 720",
            "wrong size",
            "FAIL",
            "Discard"
        ],
        [
            "8",
            "undigested ctrl",
            "single supercoiled",
            "~3.4 kb",
            "—",
            "Confirms plasmid stock identity"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0603",
                "title": "EcoRI + BamHI digest setup (6 clones)",
                "owner": "A. Marin",
                "date": "Jun 3"
            },
            {
                "ref_id": "EXP-2026-0606",
                "title": "Clones 2 + 5 Sanger sequencing setup",
                "owner": "A. Marin",
                "date": "Jun 6"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0204",
                "title": "Gel Electrophoresis SOP v2.4 (TAE / 1.2% agarose)",
                "owner": "QA Team",
                "date": "Feb 4"
            },
            {
                "ref_id": "LAD-0001",
                "title": "1 kb Plus DNA Ladder reference image + sizes",
                "owner": "NEB Docs",
                "date": "Aug 12"
            },
            {
                "ref_id": "PLM-0048",
                "title": "pUC19-GFP expected digest map",
                "owner": "Plasmid DB",
                "date": "Sep 4"
            }
        ]
    },
    "compounds": [
        {
            "name": "Agarose, molecular biology grade",
            "catalog_lot": "A9539 · Lot SLBR8814V",
            "stock_status": "In stock"
        },
        {
            "name": "50× TAE buffer (concentrate)",
            "catalog_lot": "1610773 · Lot 64158119",
            "stock_status": "In stock"
        },
        {
            "name": "SYBR Safe DNA gel stain (10,000×)",
            "catalog_lot": "S33102 · Lot 2422184",
            "stock_status": "Low"
        },
        {
            "name": "1 kb Plus DNA Ladder",
            "catalog_lot": "N3232 · Lot 10174812",
            "stock_status": "In stock"
        },
        {
            "name": "6× DNA Loading Dye (purple)",
            "catalog_lot": "B7024 · Lot 10173621",
            "stock_status": "In stock"
        }
    ],
    "storage": [
        {
            "name": "SYBR Safe stain (10,000× working)",
            "location": "4 °C · Fridge 2 · Shelf 1 · Box DNA-STAIN",
            "count": "×4"
        },
        {
            "name": "DNA ladder aliquots (50 µL working)",
            "location": "-20 °C · Freezer B-2 · Rack 1 · Box LAD-2026",
            "count": "×10"
        },
        {
            "name": "Gel image archive (TIF + raw imager output)",
            "location": "Cloud · /gels/2026-06/041/",
            "count": "×1"
        }
    ],
    "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"
            }
        ]
    }
}