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#314 Molecular Biology Complete

gDNA Extraction, HEK293 (1×10⁶ cells, DNeasy)

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gDNA HEK293 silica-column DNeasy NGS-prep

Custom ID

This template captures genomic DNA extraction from cultured HEK293 cells using a silica-membrane spin column workflow (Qiagen DNeasy Blood & Tissue Kit).

Goal: isolate high-quality genomic DNA from a 1×10⁶ HEK293 cell pellet for downstream sequencing or genotyping, targeting >50 ng/µL concentration and A260/A280 of 1.8-2.0.

Method overview: Pelleted HEK293 cells are resuspended in PBS, digested with Proteinase K in lysis buffer at 56°C for 10 minutes, ethanol-precipitated onto the silica column, washed sequentially with AW1 and AW2 buffers (centrifuged at 8,000 × g and 14,000 × g respectively), and eluted in 50 µL of AE buffer pre-warmed to 56°C. Eluate concentration is confirmed on a Qubit dsDNA HS fluorometer; purity is read on a NanoDrop spectrophotometer.

Expected: 60-100 ng/µL eluate concentration (3-5 µg total yield from 1×10⁶ HEK293 cells), A260/A280 between 1.85 and 2.0, A260/A230 above 1.8.

Last saved: 34 seconds ago

11 fields loaded from gDNA Extraction baseline (SOP v2.1)

Sample

Sample source

select

HEK293, HeLa, primary tissue, blood, FFPE

Input amount

number

1.0×10⁶ cells

Source experiment

text

EXP-2026-0412

Lysis

Lysis buffer

select

ATL, AL, custom

Proteinase K vol

number

20µL

Lysis temperature

number

56°C

Lysis duration

number

10min

Elution & QC

Elution buffer

select

AE buffer, nuclease-free H₂O

Elution volume

number

50µL

Concentration (Qubit)

number

82ng/µL

A260/A280

number

1.91

dneasy_kit_insert.pdf

342 KB

nanodrop_readings.csv

6 KB

qubit_dsDNA_HS_export.xlsx

24 KB

01

Pellet 1×10⁶ HEK293 cells (300 × g, 5 min), wash in PBS

From EXP-2026-0412

0:15

02

Resuspend pellet in 200 µL ATL, add 20 µL Proteinase K, vortex

0:05

03

Lyse at 56°C in heat block, mix occasionally

Complete lysis confirmed (clear)

0:10

04

Add 200 µL AL buffer, vortex, then 200 µL 100% ethanol

0:02

05

Transfer to DNeasy column, spin 8k × g × 1 min, discard flow

0:02

06

Wash with 500 µL AW1, spin 8k × g × 1 min, discard flow

0:02

07

Wash with 500 µL AW2, spin 14k × g × 3 min, dry membrane

Membrane dry, no residual ethanol

0:04

08

Transfer column to clean 1.5 mL tube, add 50 µL AE (56°C)

0:01

09

Incubate 1 min RT, spin 8k × g × 1 min to elute

0:02

10

QC: 1 µL on NanoDrop + 1 µL Qubit dsDNA HS, log concentration

Qubit: 79 ng/µL · NanoDrop A₂₆₀/₂₈₀ 1.91

0:08

	A	B	C	D	E	F	G
1	Sample	Vol (µL)	Conc (ng/µL)	A260/A280	A260/A230	Total yield (µg)	Notes
2	HEK293-1	50	82	1.91	2.04	4.10	Within spec
3	HEK293-2	50	74	1.88	1.96	3.70	Within spec
4	HEK293-3	50	88	1.92	2.11	4.40	Within spec
5	no-cell ctrl	50	<0.5	na	na	na	Confirms no carryover

EXP-2026-0412 HEK293 culture + pellet harvest (upstream) L. Tanaka Apr 12

EXP-2026-0418 WGS library prep (downstream, gDNA input) L. Tanaka Apr 18

SOP-0221 gDNA Extraction SOP v2.1 (mammalian cell pellet) QA Team Feb 7

KIT-0118 Qiagen DNeasy Blood & Tissue kit insert Vendor Docs Jan 14

METH-0044 NanoDrop + Qubit reading methods (gDNA QC) Methods DB Aug 30

DNeasy Blood & Tissue Kit (50 preps)

69504 · Lot 178224251

In stock

Proteinase K (>600 mAU/mL)

19131 · Lot 169033815

In stock

Ethanol, molecular grade

E7023 · Lot SHBL3927

In stock

Qubit dsDNA HS Assay Kit

Q32854 · Lot 2502184

In stock

AE elution buffer

19077 · part of kit

In stock

AW1 / AW2 wash buffers

19081 / 19072 · kit

Low

Eluate (50 µL aliquots)

-20 °C · Freezer B-1 · Drawer 3 · Box gDNA-04

×8

Source cell pellets (HEK293)

-80 °C · Freezer A-3 · Drawer 1 · Box CP-014

×3

Visibility

Tanaka LabL. Tanaka

Can write

L. TanakaDr. P. Singh

Color

Stroke width

Eraser

Use Ctrl + click to add text

{
    "experiment": {
        "id": 314,
        "custom_id": "gDNA-HEK293-2026-04",
        "title": "gDNA Extraction, HEK293 (1×10⁶ cells, DNeasy)",
        "template": "DNA Extraction",
        "template_version": "v3.2",
        "category": "Molecular Biology",
        "status": "Complete",
        "started_on": "2026-06-06",
        "tags": [
            "gDNA",
            "HEK293",
            "silica-column",
            "DNeasy",
            "NGS-prep"
        ]
    },
    "main_text": "This template captures genomic DNA extraction from cultured HEK293 cells using a silica-membrane spin column workflow (Qiagen DNeasy Blood & Tissue Kit).\nGoal: isolate high-quality genomic DNA from a 1×10⁶ HEK293 cell pellet for downstream sequencing or genotyping, targeting >50 ng/µL concentration and A260/A280 of 1.8-2.0.\nMethod overview: Pelleted HEK293 cells are resuspended in PBS, digested with Proteinase K in lysis buffer at 56°C for 10 minutes, ethanol-precipitated onto the silica column, washed sequentially with AW1 and AW2 buffers (centrifuged at 8,000 × g and 14,000 × g respectively), and eluted in 50 µL of AE buffer pre-warmed to 56°C. Eluate concentration is confirmed on a Qubit dsDNA HS fluorometer; purity is read on a NanoDrop spectrophotometer.\nExpected: 60-100 ng/µL eluate concentration (3-5 µg total yield from 1×10⁶ HEK293 cells), A260/A280 between 1.85 and 2.0, A260/A230 above 1.8.",
    "extra_fields": {
        "Sample": {
            "Sample source": {
                "type": "select",
                "value": "HEK293, HeLa, primary tissue, blood, FFPE"
            },
            "Input amount": {
                "type": "number",
                "value": "1.0 ×10⁶ cells"
            },
            "Source experiment": {
                "type": "text",
                "value": "EXP-2026-0412"
            }
        },
        "Lysis": {
            "Lysis buffer": {
                "type": "select",
                "value": "ATL, AL, custom"
            },
            "Proteinase K vol": {
                "type": "number",
                "value": "20 µL"
            },
            "Lysis temperature": {
                "type": "number",
                "value": "56 °C"
            },
            "Lysis duration": {
                "type": "number",
                "value": "10 min"
            }
        },
        "Elution & QC": {
            "Elution buffer": {
                "type": "select",
                "value": "AE buffer, nuclease-free H₂O"
            },
            "Elution volume": {
                "type": "number",
                "value": "50 µL"
            },
            "Concentration (Qubit)": {
                "type": "number",
                "value": "82 ng/µL"
            },
            "A260/A280": {
                "type": "number",
                "value": "1.91"
            }
        }
    },
    "attached_files": [
        {
            "filename": "dneasy_kit_insert.pdf",
            "type": "pdf",
            "size": "342 KB"
        },
        {
            "filename": "nanodrop_readings.csv",
            "type": "csv",
            "size": "6 KB"
        },
        {
            "filename": "qubit_dsDNA_HS_export.xlsx",
            "type": "xlsx",
            "size": "24 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Pellet 1×10⁶ HEK293 cells (300 × g, 5 min), wash in PBS",
            "duration": "0:15",
            "complete": true,
            "note": "From EXP-2026-0412"
        },
        {
            "n": 2,
            "description": "Resuspend pellet in 200 µL ATL, add 20 µL Proteinase K, vortex",
            "duration": "0:05",
            "complete": true
        },
        {
            "n": 3,
            "description": "Lyse at 56°C in heat block, mix occasionally",
            "duration": "0:10",
            "complete": true,
            "note": "Complete lysis confirmed (clear)"
        },
        {
            "n": 4,
            "description": "Add 200 µL AL buffer, vortex, then 200 µL 100% ethanol",
            "duration": "0:02",
            "complete": true
        },
        {
            "n": 5,
            "description": "Transfer to DNeasy column, spin 8k × g × 1 min, discard flow",
            "duration": "0:02",
            "complete": true
        },
        {
            "n": 6,
            "description": "Wash with 500 µL AW1, spin 8k × g × 1 min, discard flow",
            "duration": "0:02",
            "complete": true
        },
        {
            "n": 7,
            "description": "Wash with 500 µL AW2, spin 14k × g × 3 min, dry membrane",
            "duration": "0:04",
            "complete": true,
            "note": "Membrane dry, no residual ethanol"
        },
        {
            "n": 8,
            "description": "Transfer column to clean 1.5 mL tube, add 50 µL AE (56°C)",
            "duration": "0:01",
            "complete": true
        },
        {
            "n": 9,
            "description": "Incubate 1 min RT, spin 8k × g × 1 min to elute",
            "duration": "0:02",
            "complete": true
        },
        {
            "n": 10,
            "description": "QC: 1 µL on NanoDrop + 1 µL Qubit dsDNA HS, log concentration",
            "duration": "0:08",
            "complete": true,
            "note": "Qubit: 79 ng/µL · NanoDrop A₂₆₀/₂₈₀ 1.91"
        }
    ],
    "spreadsheet": [
        [
            "Sample",
            "Vol (µL)",
            "Conc (ng/µL)",
            "A260/A280",
            "A260/A230",
            "Total yield (µg)",
            "Notes"
        ],
        [
            "HEK293-1",
            "50",
            "82",
            "1.91",
            "2.04",
            "4.10",
            "Within spec"
        ],
        [
            "HEK293-2",
            "50",
            "74",
            "1.88",
            "1.96",
            "3.70",
            "Within spec"
        ],
        [
            "HEK293-3",
            "50",
            "88",
            "1.92",
            "2.11",
            "4.40",
            "Within spec"
        ],
        [
            "no-cell ctrl",
            "50",
            "<0.5",
            "na",
            "na",
            "na",
            "Confirms no carryover"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0412",
                "title": "HEK293 culture + pellet harvest (upstream)",
                "owner": "L. Tanaka",
                "date": "Apr 12"
            },
            {
                "ref_id": "EXP-2026-0418",
                "title": "WGS library prep (downstream, gDNA input)",
                "owner": "L. Tanaka",
                "date": "Apr 18"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0221",
                "title": "gDNA Extraction SOP v2.1 (mammalian cell pellet)",
                "owner": "QA Team",
                "date": "Feb 7"
            },
            {
                "ref_id": "KIT-0118",
                "title": "Qiagen DNeasy Blood & Tissue kit insert",
                "owner": "Vendor Docs",
                "date": "Jan 14"
            },
            {
                "ref_id": "METH-0044",
                "title": "NanoDrop + Qubit reading methods (gDNA QC)",
                "owner": "Methods DB",
                "date": "Aug 30"
            }
        ]
    },
    "compounds": [
        {
            "name": "DNeasy Blood & Tissue Kit (50 preps)",
            "catalog_lot": "69504 · Lot 178224251",
            "stock_status": "In stock"
        },
        {
            "name": "Proteinase K (>600 mAU/mL)",
            "catalog_lot": "19131 · Lot 169033815",
            "stock_status": "In stock"
        },
        {
            "name": "Ethanol, molecular grade",
            "catalog_lot": "E7023 · Lot SHBL3927",
            "stock_status": "In stock"
        },
        {
            "name": "Qubit dsDNA HS Assay Kit",
            "catalog_lot": "Q32854 · Lot 2502184",
            "stock_status": "In stock"
        },
        {
            "name": "AE elution buffer",
            "catalog_lot": "19077 · part of kit",
            "stock_status": "In stock"
        },
        {
            "name": "AW1 / AW2 wash buffers",
            "catalog_lot": "19081 / 19072 · kit",
            "stock_status": "Low"
        }
    ],
    "storage": [
        {
            "name": "Eluate (50 µL aliquots)",
            "location": "-20 °C · Freezer B-1 · Drawer 3 · Box gDNA-04",
            "count": "×8"
        },
        {
            "name": "Source cell pellets (HEK293)",
            "location": "-80 °C · Freezer A-3 · Drawer 1 · Box CP-014",
            "count": "×3"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "Tanaka Lab"
            },
            {
                "type": "Person",
                "name": "L. Tanaka"
            }
        ],
        "can_write": [
            {
                "type": "Person",
                "name": "L. Tanaka"
            },
            {
                "type": "Person",
                "name": "Dr. P. Singh"
            }
        ]
    }
}