Transfection Calculator (PEI & Lipofectamine)

What this is for: DNA, PEI and Lipofectamine 3000/P3000 volumes per vessel format and run totals, including co-transfection plasmid shares.

The formula: reagent (µL) = DNA per vessel (µg) × ratio (for 1 mg/mL PEI stock)

How to use:

In the PEI section, pick the vessel format and enter the number of vessels, overage, and PEI ratios A and B.
If co-transfecting, enter each plasmid's share percentage and stock concentration in ng/µL.
Read the per-vessel PEI volumes, plasmid volumes, and the totals for all vessels with overage.
In the Lipofectamine 3000 section, pick the vessel format, number of vessels, overage, and the Lipofectamine and P3000 rates, then read the per-vessel and total volumes.

PEI Transfection Calculator (incl. co-transfection)

Pick a vessel — standard media volume, DNA amount and seeding density fill in from the reference table. PEI stock is 1 mg/mL, so µL of PEI = µg DNA × ratio. This lab's standard ratio is 1:6 (DNA:PEI); 1:3 is shown alongside.

Vessel format

Number of vessels

Overage%

PEI ratio A (DNA:PEI)

PEI ratio B (DNA:PEI) — lab standard

DNA per vessel (µg)

Media per vessel (mL)

Cells to seed per vessel

Diluent per mix (µL)

PEI per vessel at ratio A (µL)

PEI per vessel at ratio B (µL)

Co-transfection — plasmid shares (per vessel)

Plasmid 1 (e.g. transfer plasmid) — Share%

Plasmid 1 (e.g. transfer plasmid) — Stockng/µL

Plasmid 1 (e.g. transfer plasmid) — µL per vessel

Plasmid 2 (e.g. packaging) — Share%

Plasmid 2 (e.g. packaging) — Stockng/µL

Plasmid 2 (e.g. packaging) — µL per vessel

Plasmid 3 (e.g. envelope) — Share%

Plasmid 3 (e.g. envelope) — Stockng/µL

Plasmid 3 (e.g. envelope) — µL per vessel

Plasmid 4 — Share%

Plasmid 4 — Stockng/µL

Plasmid 4 — µL per vessel

Total plasmid volume per vessel (µL)

Totals for all vessels + overage

Total DNA (µg)

Total PEI at ratio B (µL)

DNA-mix diluent (Opti-MEM) total (µL)

PEI-mix diluent (Opti-MEM) total (µL)

Method: dilute DNA in diluent; dilute PEI separately; rest 5 min; combine, incubate 15–20 min, add to cells in media.

Vessel format	Media (mL)	DNA per vessel (µg)	Cells to seed per vessel	Diluent per mix (µL)
96-well	0.1	0.1	20000	10
24-well	0.5	0.5	100000	25
12-well	1	1	200000	50
6-well	2	2.5	500000	100
T25	5	6	1500000	250
T75	15	16	5000000	500

Lipofectamine 3000 Transfection Calculator

Vessel values come from the reference table on the PEI sheet. Defaults: Lipofectamine 3000 at 1.5 µL per µg DNA and P3000 at 2 µL per µg DNA — both editable. DNA and P3000 go in one diluent tube; Lipofectamine in a second; combine 1:1.

Vessel format

Number of vessels

Overage%

Lipofectamine 3000 (µL per µg DNA)

P3000 (µL per µg DNA)

DNA per vessel (µg)

Diluent per mix (µL)

Lipofectamine 3000 per vessel (µL)

P3000 per vessel (µL)

Total DNA (µg)

Total Lipofectamine 3000 (µL)

Total P3000 (µL)

Total diluent, both mixes (µL)

Prefer to work offline or keep a copy? The download link is right below.
Download the Transfection Calculator (PEI & Lipofectamine) workbook (.xlsx)

Notes & limits (from the workbook): PEI stock is 1 mg/mL, so µL of PEI = µg DNA × ratio. This lab’s standard ratio is 1:6 (DNA:PEI); 1:3 is shown alongside. Vessel values come from the reference table — edit it in the workbook if your lab’s values differ. Lipofectamine defaults: 1.5 µL per µg DNA and P3000 at 2 µL per µg DNA, both editable. Method: dilute DNA in diluent; dilute PEI separately; rest 5 min; combine, incubate 15–20 min, add to cells in media.