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#314 Molecular Biology Running

Transformation, pUC19-GFP into DH5α

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transformation cloning DH5α pUC19 amp-selection

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This template captures heat-shock transformation of chemically competent E. coli DH5α with the pUC19-GFP cloning vector.

Goal: introduce the pUC19-GFP plasmid into DH5α for plasmid amplification and downstream sequencing verification, with ampicillin selection on LB agar.

Method overview: Competent DH5α cells are thawed on ice, 50 ng of plasmid DNA is added, cells are incubated on ice for 30 minutes, heat-shocked at 42°C for 45 seconds, returned to ice for 2 minutes, recovered in 950 µL of SOC at 37°C with shaking for 1 hour, then plated on LB + ampicillin (100 µg/mL) and incubated overnight at 37°C. Colonies are counted the next morning and selected colonies are picked for sequencing verification and glycerol stock preparation.

Expected: 50-500 colonies on the experimental plate (efficiency ~10⁶ CFU/µg with DH5α and high-copy vector), zero colonies on the no-DNA control plate.

Last saved: 34 seconds ago

11 fields loaded from Bacterial Transformation baseline (Cloning SOP v1.3)

Sample

Competent strain

select

DH5α, TOP10, BL21(DE3), Stbl3, JM109

Plasmid

text

pUC19-GFP

DNA input amount

number

50ng

Transformation

Ice incubation

number

30min

Heat shock temp

number

42°C

Heat shock duration

number

45s

Post-shock on ice

number

2min

Recovery & Plating

Recovery medium

select

SOC, LB

Recovery time

number

60min

Plating volume

number

100µL

Selection antibiotic

text

Amp 100 µg/mL

pUC19-GFP_map.pdf

212 KB

plate_count_photo.png

1.8 MB

colony_pick_log.csv

14 KB

01

Pre-warm SOC + LB+Amp plates at 37°C

0:15

02

Thaw competent DH5α aliquot on ice (~10 min)

0:10

03

Add 50 ng pUC19-GFP to 50 µL cells, flick to mix

No-DNA ctrl tube in parallel

0:02

04

Incubate on ice 30 minutes

0:30

05

Heat shock 42°C × 45 s in water bath, return to ice 2 min

Recorded at 10:14 by AM

0:03

06

Add 950 µL SOC, recover at 37°C with shaking

225 rpm

1:00

07

Plate 100 µL on LB+Amp 100 (and 100 µL on no-DNA ctrl)

Spread with beads

0:10

08

Invert plates, incubate 37°C overnight (16-18 h)

17:00

09

Count colonies, calculate transformation efficiency

237 cfu / plate

0:20

10

Pick 8 colonies into LB+Amp for o/n culture + sequencing

Awaiting Mon AM

0:15

	A	B	C	D	E	F	G
1	Plate	Condition	DNA (ng)	Plated (µL)	Colonies	CFU/µg	Notes
2	1	+DNA (pUC19-GFP)	50	100	237	4.74×10⁶	Good lawn-free spread
3	2	+DNA, 1:10 dilute	50	10	26	5.20×10⁶	Confirms plate 1 count
4	3	no-DNA ctrl	0	100	0	0	Clean, contamination ruled out
5	4	no-Amp ctrl	50	100	lawn	na	Cells viable

EXP-2026-0419 pUC19-GFP plasmid miniprep (upstream) A. Marin Apr 19

EXP-2026-0421 Colony PCR verification of pUC19-GFP clones A. Marin Apr 21

SOP-0117 Bacterial Transformation Standard Protocol v1.3 QA Team Jan 22

PLM-0048 pUC19-GFP plasmid map and sequence Plasmid DB Sep 4

STR-0019 DH5α competent cell prep & validation log Strain DB Mar 1

DH5α chemically competent E. coli

18265017 · Lot 2521403

In stock

pUC19-GFP plasmid prep (60 ng/µL)

PLM-0048 · Prep 2026-04

In stock

Ampicillin sodium salt (100 mg/mL)

A9518 · Lot SLBP1247V

In stock

SOC medium

15544034 · Lot 2401821

In stock

LB agar + 100 µg/mL Amp plates

Lab-poured · Batch 04-08

Low

X-gal / IPTG (for blue/white screen)

R0941 · Lot 26

In stock

DH5α competent aliquots (50 µL)

-80 °C · Freezer A-3 · Drawer 2 · Box CC-DH5α-04

×42

pUC19-GFP plasmid stock (60 ng/µL)

-20 °C · Freezer B-1 · Rack 4 · Tube P-048

×1

Glycerol stocks (transformed clones)

-80 °C · Freezer A-3 · Drawer 4 · Box GS-2026-04

×8 pending

Visibility

Marin LabA. Marin

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A. MarinDr. J. Park

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{
    "experiment": {
        "id": 314,
        "custom_id": "TXF-pUC19-2026-04",
        "title": "Transformation, pUC19-GFP into DH5α",
        "template": "Bacterial Transformation",
        "template_version": "v3.2",
        "category": "Molecular Biology",
        "status": "Running",
        "started_on": "2026-05-29",
        "tags": [
            "transformation",
            "cloning",
            "DH5α",
            "pUC19",
            "amp-selection"
        ]
    },
    "main_text": "This template captures heat-shock transformation of chemically competent E. coli DH5α with the pUC19-GFP cloning vector.\nGoal: introduce the pUC19-GFP plasmid into DH5α for plasmid amplification and downstream sequencing verification, with ampicillin selection on LB agar.\nMethod overview: Competent DH5α cells are thawed on ice, 50 ng of plasmid DNA is added, cells are incubated on ice for 30 minutes, heat-shocked at 42°C for 45 seconds, returned to ice for 2 minutes, recovered in 950 µL of SOC at 37°C with shaking for 1 hour, then plated on LB + ampicillin (100 µg/mL) and incubated overnight at 37°C. Colonies are counted the next morning and selected colonies are picked for sequencing verification and glycerol stock preparation.\nExpected: 50-500 colonies on the experimental plate (efficiency ~10⁶ CFU/µg with DH5α and high-copy vector), zero colonies on the no-DNA control plate.",
    "extra_fields": {
        "Sample": {
            "Competent strain": {
                "type": "select",
                "value": "DH5α, TOP10, BL21(DE3), Stbl3, JM109"
            },
            "Plasmid": {
                "type": "text",
                "value": "pUC19-GFP"
            },
            "DNA input amount": {
                "type": "number",
                "value": "50 ng"
            }
        },
        "Transformation": {
            "Ice incubation": {
                "type": "number",
                "value": "30 min"
            },
            "Heat shock temp": {
                "type": "number",
                "value": "42 °C"
            },
            "Heat shock duration": {
                "type": "number",
                "value": "45 s"
            },
            "Post-shock on ice": {
                "type": "number",
                "value": "2 min"
            }
        },
        "Recovery & Plating": {
            "Recovery medium": {
                "type": "select",
                "value": "SOC, LB"
            },
            "Recovery time": {
                "type": "number",
                "value": "60 min"
            },
            "Plating volume": {
                "type": "number",
                "value": "100 µL"
            },
            "Selection antibiotic": {
                "type": "text",
                "value": "Amp 100 µg/mL"
            }
        }
    },
    "attached_files": [
        {
            "filename": "pUC19-GFP_map.pdf",
            "type": "pdf",
            "size": "212 KB"
        },
        {
            "filename": "plate_count_photo.png",
            "type": "png",
            "size": "1.8 MB"
        },
        {
            "filename": "colony_pick_log.csv",
            "type": "csv",
            "size": "14 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Pre-warm SOC + LB+Amp plates at 37°C",
            "duration": "0:15",
            "complete": true
        },
        {
            "n": 2,
            "description": "Thaw competent DH5α aliquot on ice (~10 min)",
            "duration": "0:10",
            "complete": true
        },
        {
            "n": 3,
            "description": "Add 50 ng pUC19-GFP to 50 µL cells, flick to mix",
            "duration": "0:02",
            "complete": true,
            "note": "No-DNA ctrl tube in parallel"
        },
        {
            "n": 4,
            "description": "Incubate on ice 30 minutes",
            "duration": "0:30",
            "complete": true
        },
        {
            "n": 5,
            "description": "Heat shock 42°C × 45 s in water bath, return to ice 2 min",
            "duration": "0:03",
            "complete": true,
            "note": "Recorded at 10:14 by AM"
        },
        {
            "n": 6,
            "description": "Add 950 µL SOC, recover at 37°C with shaking",
            "duration": "1:00",
            "complete": true,
            "note": "225 rpm"
        },
        {
            "n": 7,
            "description": "Plate 100 µL on LB+Amp 100 (and 100 µL on no-DNA ctrl)",
            "duration": "0:10",
            "complete": true,
            "note": "Spread with beads"
        },
        {
            "n": 8,
            "description": "Invert plates, incubate 37°C overnight (16-18 h)",
            "duration": "17:00",
            "complete": true
        },
        {
            "n": 9,
            "description": "Count colonies, calculate transformation efficiency",
            "duration": "0:20",
            "complete": true,
            "note": "237 cfu / plate"
        },
        {
            "n": 10,
            "description": "Pick 8 colonies into LB+Amp for o/n culture + sequencing",
            "duration": "0:15",
            "complete": false,
            "note": "Awaiting Mon AM"
        }
    ],
    "spreadsheet": [
        [
            "Plate",
            "Condition",
            "DNA (ng)",
            "Plated (µL)",
            "Colonies",
            "CFU/µg",
            "Notes"
        ],
        [
            "1",
            "+DNA (pUC19-GFP)",
            "50",
            "100",
            "237",
            "4.74×10⁶",
            "Good lawn-free spread"
        ],
        [
            "2",
            "+DNA, 1:10 dilute",
            "50",
            "10",
            "26",
            "5.20×10⁶",
            "Confirms plate 1 count"
        ],
        [
            "3",
            "no-DNA ctrl",
            "0",
            "100",
            "0",
            "0",
            "Clean, contamination ruled out"
        ],
        [
            "4",
            "no-Amp ctrl",
            "50",
            "100",
            "lawn",
            "na",
            "Cells viable"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0419",
                "title": "pUC19-GFP plasmid miniprep (upstream)",
                "owner": "A. Marin",
                "date": "Apr 19"
            },
            {
                "ref_id": "EXP-2026-0421",
                "title": "Colony PCR verification of pUC19-GFP clones",
                "owner": "A. Marin",
                "date": "Apr 21"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0117",
                "title": "Bacterial Transformation Standard Protocol v1.3",
                "owner": "QA Team",
                "date": "Jan 22"
            },
            {
                "ref_id": "PLM-0048",
                "title": "pUC19-GFP plasmid map and sequence",
                "owner": "Plasmid DB",
                "date": "Sep 4"
            },
            {
                "ref_id": "STR-0019",
                "title": "DH5α competent cell prep & validation log",
                "owner": "Strain DB",
                "date": "Mar 1"
            }
        ]
    },
    "compounds": [
        {
            "name": "DH5α chemically competent E. coli",
            "catalog_lot": "18265017 · Lot 2521403",
            "stock_status": "In stock"
        },
        {
            "name": "pUC19-GFP plasmid prep (60 ng/µL)",
            "catalog_lot": "PLM-0048 · Prep 2026-04",
            "stock_status": "In stock"
        },
        {
            "name": "Ampicillin sodium salt (100 mg/mL)",
            "catalog_lot": "A9518 · Lot SLBP1247V",
            "stock_status": "In stock"
        },
        {
            "name": "SOC medium",
            "catalog_lot": "15544034 · Lot 2401821",
            "stock_status": "In stock"
        },
        {
            "name": "LB agar + 100 µg/mL Amp plates",
            "catalog_lot": "Lab-poured · Batch 04-08",
            "stock_status": "Low"
        },
        {
            "name": "X-gal / IPTG (for blue/white screen)",
            "catalog_lot": "R0941 · Lot 26",
            "stock_status": "In stock"
        }
    ],
    "storage": [
        {
            "name": "DH5α competent aliquots (50 µL)",
            "location": "-80 °C · Freezer A-3 · Drawer 2 · Box CC-DH5α-04",
            "count": "×42"
        },
        {
            "name": "pUC19-GFP plasmid stock (60 ng/µL)",
            "location": "-20 °C · Freezer B-1 · Rack 4 · Tube P-048",
            "count": "×1"
        },
        {
            "name": "Glycerol stocks (transformed clones)",
            "location": "-80 °C · Freezer A-3 · Drawer 4 · Box GS-2026-04",
            "count": "×8 pending"
        }
    ],
    "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"
            }
        ]
    }
}