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#314 Molecular Biology Complete

PCR, GAPDH amplification (498 bp) from gDNA

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PCR GAPDH Q5-polymerase primer-design amplification

Custom ID

This template captures a standard endpoint PCR amplification of a 498 bp fragment of GAPDH from human genomic DNA, used as a positive control for downstream cloning, sequencing, or colony screening workflows.

Goal: amplify a clean, single-band 498 bp GAPDH product from 1 ng of HeLa genomic DNA template, verify by 1% agarose gel, and confirm absence of non-specific amplification in the no-template control (NTC).

Method overview: Reactions are set up in 25 µL volumes with Q5 High-Fidelity polymerase, 0.5 µM each primer (GAPDH-F / GAPDH-R), 200 µM dNTPs, and 1 ng input genomic DNA. Cycling: 98°C 30 s initial denaturation; 30 cycles of 98°C 10 s, 58°C 30 s, 72°C 30 s; 72°C 2 min final extension. Products run on 1% agarose with SYBR Safe at 100 V for 35 minutes alongside a 1 kb DNA ladder. Imaging on UV transilluminator.

Expected: Single sharp band at 498 bp in sample lanes (HeLa gDNA × 5 replicates). No band in NTC lane. Ladder reference confirms size assignment.

Last saved: 34 seconds ago

11 fields loaded from PCR Standard baseline (SOP v2.1)

Template

Template source

select

gDNA, cDNA, plasmid, colony

Input amount

number

1.0ng

Source reference

text

EXP-2026-0412 (HeLa gDNA prep)

Reaction

Polymerase

select

Q5 HiFi, Phusion, Taq, KAPA HiFi

Reaction volume

number

25µL

Primer conc

number

0.5µM each

dNTP conc

number

200µM each

Cycling

Annealing temp

number

58°C

Extension time

number

30s

Cycle count

number

30

Expected size

number

498bp

GAPDH_primer_design.pdf

184 KB

agarose_gel_image.tif

2.6 MB

band_intensity.csv

8 KB

01

Thaw primer working stocks, polymerase, dNTPs on ice

Recorded at 09:14 by LM

0:10

02

Prepare 6-reaction master mix (5 samples + NTC) with 10% excess

Q5 1× buffer + primers + dNTPs

0:10

03

Aliquot 23 µL master mix into 6 PCR tubes

0:03

04

Add 2 µL template (HeLa gDNA, 0.5 ng/µL) to sample tubes; H₂O to NTC

0:03

05

Brief spin (1k × 30s) to collect contents

0:01

06

Load thermocycler: 98°C 30 s, [98°C 10 s · 58°C 30 s · 72°C 30 s] ×30, 72°C 2 min

C1000 Touch · run ID 2026-0604-09:30

1:20

07

Pour 1% agarose gel with SYBR Safe (50 mL TAE × 0.5 g agarose × 5 µL SYBR)

0:30

08

Mix 5 µL PCR product + 1 µL loading dye, load alongside 5 µL of 1 kb ladder

0:08

09

Run gel: 100 V × 35 min until dye front 2/3 down

0:35

10

Image on UV transilluminator, save TIF + record band intensities

498 bp band in lanes 1-3 + 5; lane 4 faint; NTC clean

0:05

	A	B	C	D	E	F
1	Lane	Sample	Expected (bp)	Observed (bp)	Intensity	Notes
2	1	HeLa-gDNA-1	498	~498	strong	Clean single band
3	2	HeLa-gDNA-2	498	~498	strong	Clean single band
4	3	HeLa-gDNA-3	498	~498	strong	Clean single band
5	4	HeLa-gDNA-4	498	~498	faint	Recheck template — possible dilution error
6	5	HeLa-gDNA-5	498	~498	strong	Clean single band
7	NTC	no template	—	no band	—	Confirms no contamination
8	M	1 kb ladder	reference	reference	ladder	500, 250, 1000 bands visible

EXP-2026-0412 HeLa gDNA extraction (template source) L. Madison Apr 12

EXP-2026-0603 GAPDH primer Tm validation gradient L. Madison Jun 3

SOP-0218 PCR Standard Protocol v2.1 (Q5 HiFi reactions) QA Team Feb 8

PRIMER-018 GAPDH-F / GAPDH-R primer pair (498 bp product) Primer DB Jan 22

SOP-0301 Agarose Gel Imaging SOP QA Team Mar 1

Q5 High-Fidelity DNA Polymerase (2× Master Mix)

M0492 · Lot 10174924

In stock

GAPDH forward primer (10 µM working)

IDT-018a · Lot 20240118

In stock

GAPDH reverse primer (10 µM working)

IDT-018b · Lot 20240118

In stock

dNTP mix (10 mM each)

N0447 · Lot 10172841

In stock

1 kb DNA Ladder

N3232 · Lot 10174812

In stock

SYBR Safe DNA Gel Stain (10,000×)

S33102 · Lot 2422184

Low

Primer working stocks (10 µM)

-20 °C · Freezer B-2 · Rack 1 · Box PR-018

×2

Q5 polymerase aliquots (100 µL working)

-20 °C · Freezer B-2 · Rack 2 · Box ENZ-04

×6

PCR products (post-reaction archive)

-20 °C · Freezer C-1 · Drawer 1 · Box PCR-2026-06

×6

Visibility

Madison LabL. Madison

Can write

L. MadisonDr. J. Park

Color

Stroke width

Eraser

Use Ctrl + click to add text

{
    "experiment": {
        "id": 314,
        "custom_id": "PCR-GAPDH-2026-06",
        "title": "PCR, GAPDH amplification (498 bp) from gDNA",
        "template": "PCR",
        "template_version": "v3.2",
        "category": "Molecular Biology",
        "status": "Complete",
        "started_on": "2026-07-15",
        "tags": [
            "PCR",
            "GAPDH",
            "Q5-polymerase",
            "primer-design",
            "amplification"
        ]
    },
    "main_text": "This template captures a standard endpoint PCR amplification of a 498 bp fragment of GAPDH from human genomic DNA, used as a positive control for downstream cloning, sequencing, or colony screening workflows.\nGoal: amplify a clean, single-band 498 bp GAPDH product from 1 ng of HeLa genomic DNA template, verify by 1% agarose gel, and confirm absence of non-specific amplification in the no-template control (NTC).\nMethod overview: Reactions are set up in 25 µL volumes with Q5 High-Fidelity polymerase, 0.5 µM each primer (GAPDH-F / GAPDH-R), 200 µM dNTPs, and 1 ng input genomic DNA. Cycling: 98°C 30 s initial denaturation; 30 cycles of 98°C 10 s, 58°C 30 s, 72°C 30 s; 72°C 2 min final extension. Products run on 1% agarose with SYBR Safe at 100 V for 35 minutes alongside a 1 kb DNA ladder. Imaging on UV transilluminator.\nExpected: Single sharp band at 498 bp in sample lanes (HeLa gDNA × 5 replicates). No band in NTC lane. Ladder reference confirms size assignment.",
    "extra_fields": {
        "Template": {
            "Template source": {
                "type": "select",
                "value": "gDNA, cDNA, plasmid, colony"
            },
            "Input amount": {
                "type": "number",
                "value": "1.0 ng"
            },
            "Source reference": {
                "type": "text",
                "value": "EXP-2026-0412 (HeLa gDNA prep)"
            }
        },
        "Reaction": {
            "Polymerase": {
                "type": "select",
                "value": "Q5 HiFi, Phusion, Taq, KAPA HiFi"
            },
            "Reaction volume": {
                "type": "number",
                "value": "25 µL"
            },
            "Primer conc": {
                "type": "number",
                "value": "0.5 µM each"
            },
            "dNTP conc": {
                "type": "number",
                "value": "200 µM each"
            }
        },
        "Cycling": {
            "Annealing temp": {
                "type": "number",
                "value": "58 °C"
            },
            "Extension time": {
                "type": "number",
                "value": "30 s"
            },
            "Cycle count": {
                "type": "number",
                "value": "30"
            },
            "Expected size": {
                "type": "number",
                "value": "498 bp"
            }
        }
    },
    "attached_files": [
        {
            "filename": "GAPDH_primer_design.pdf",
            "type": "pdf",
            "size": "184 KB"
        },
        {
            "filename": "agarose_gel_image.tif",
            "type": "png",
            "size": "2.6 MB"
        },
        {
            "filename": "band_intensity.csv",
            "type": "csv",
            "size": "8 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Thaw primer working stocks, polymerase, dNTPs on ice",
            "duration": "0:10",
            "complete": true,
            "note": "Recorded at 09:14 by LM"
        },
        {
            "n": 2,
            "description": "Prepare 6-reaction master mix (5 samples + NTC) with 10% excess",
            "duration": "0:10",
            "complete": true,
            "note": "Q5 1× buffer + primers + dNTPs"
        },
        {
            "n": 3,
            "description": "Aliquot 23 µL master mix into 6 PCR tubes",
            "duration": "0:03",
            "complete": true
        },
        {
            "n": 4,
            "description": "Add 2 µL template (HeLa gDNA, 0.5 ng/µL) to sample tubes; H₂O to NTC",
            "duration": "0:03",
            "complete": true
        },
        {
            "n": 5,
            "description": "Brief spin (1k × 30s) to collect contents",
            "duration": "0:01",
            "complete": true
        },
        {
            "n": 6,
            "description": "Load thermocycler: 98°C 30 s, [98°C 10 s · 58°C 30 s · 72°C 30 s] ×30, 72°C 2 min",
            "duration": "1:20",
            "complete": true,
            "note": "C1000 Touch · run ID 2026-0604-09:30"
        },
        {
            "n": 7,
            "description": "Pour 1% agarose gel with SYBR Safe (50 mL TAE × 0.5 g agarose × 5 µL SYBR)",
            "duration": "0:30",
            "complete": true
        },
        {
            "n": 8,
            "description": "Mix 5 µL PCR product + 1 µL loading dye, load alongside 5 µL of 1 kb ladder",
            "duration": "0:08",
            "complete": true
        },
        {
            "n": 9,
            "description": "Run gel: 100 V × 35 min until dye front 2/3 down",
            "duration": "0:35",
            "complete": true
        },
        {
            "n": 10,
            "description": "Image on UV transilluminator, save TIF + record band intensities",
            "duration": "0:05",
            "complete": true,
            "note": "498 bp band in lanes 1-3 + 5; lane 4 faint; NTC clean"
        }
    ],
    "spreadsheet": [
        [
            "Lane",
            "Sample",
            "Expected (bp)",
            "Observed (bp)",
            "Intensity",
            "Notes"
        ],
        [
            "1",
            "HeLa-gDNA-1",
            "498",
            "~498",
            "strong",
            "Clean single band"
        ],
        [
            "2",
            "HeLa-gDNA-2",
            "498",
            "~498",
            "strong",
            "Clean single band"
        ],
        [
            "3",
            "HeLa-gDNA-3",
            "498",
            "~498",
            "strong",
            "Clean single band"
        ],
        [
            "4",
            "HeLa-gDNA-4",
            "498",
            "~498",
            "faint",
            "Recheck template — possible dilution error"
        ],
        [
            "5",
            "HeLa-gDNA-5",
            "498",
            "~498",
            "strong",
            "Clean single band"
        ],
        [
            "NTC",
            "no template",
            "—",
            "no band",
            "—",
            "Confirms no contamination"
        ],
        [
            "M",
            "1 kb ladder",
            "reference",
            "reference",
            "ladder",
            "500, 250, 1000 bands visible"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0412",
                "title": "HeLa gDNA extraction (template source)",
                "owner": "L. Madison",
                "date": "Apr 12"
            },
            {
                "ref_id": "EXP-2026-0603",
                "title": "GAPDH primer Tm validation gradient",
                "owner": "L. Madison",
                "date": "Jun 3"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0218",
                "title": "PCR Standard Protocol v2.1 (Q5 HiFi reactions)",
                "owner": "QA Team",
                "date": "Feb 8"
            },
            {
                "ref_id": "PRIMER-018",
                "title": "GAPDH-F / GAPDH-R primer pair (498 bp product)",
                "owner": "Primer DB",
                "date": "Jan 22"
            },
            {
                "ref_id": "SOP-0301",
                "title": "Agarose Gel Imaging SOP",
                "owner": "QA Team",
                "date": "Mar 1"
            }
        ]
    },
    "compounds": [
        {
            "name": "Q5 High-Fidelity DNA Polymerase (2× Master Mix)",
            "catalog_lot": "M0492 · Lot 10174924",
            "stock_status": "In stock"
        },
        {
            "name": "GAPDH forward primer (10 µM working)",
            "catalog_lot": "IDT-018a · Lot 20240118",
            "stock_status": "In stock"
        },
        {
            "name": "GAPDH reverse primer (10 µM working)",
            "catalog_lot": "IDT-018b · Lot 20240118",
            "stock_status": "In stock"
        },
        {
            "name": "dNTP mix (10 mM each)",
            "catalog_lot": "N0447 · Lot 10172841",
            "stock_status": "In stock"
        },
        {
            "name": "1 kb DNA Ladder",
            "catalog_lot": "N3232 · Lot 10174812",
            "stock_status": "In stock"
        },
        {
            "name": "SYBR Safe DNA Gel Stain (10,000×)",
            "catalog_lot": "S33102 · Lot 2422184",
            "stock_status": "Low"
        }
    ],
    "storage": [
        {
            "name": "Primer working stocks (10 µM)",
            "location": "-20 °C · Freezer B-2 · Rack 1 · Box PR-018",
            "count": "×2"
        },
        {
            "name": "Q5 polymerase aliquots (100 µL working)",
            "location": "-20 °C · Freezer B-2 · Rack 2 · Box ENZ-04",
            "count": "×6"
        },
        {
            "name": "PCR products (post-reaction archive)",
            "location": "-20 °C · Freezer C-1 · Drawer 1 · Box PCR-2026-06",
            "count": "×6"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "Madison Lab"
            },
            {
                "type": "Person",
                "name": "L. Madison"
            }
        ],
        "can_write": [
            {
                "type": "Person",
                "name": "L. Madison"
            },
            {
                "type": "Person",
                "name": "Dr. J. Park"
            }
        ]
    }
}