Does anyone have success transfecting plasmid DNA into BxPC3 cells using Lipofectamine 2000/3000 and could share a protocol? Waiting for my plasmid to arrive and would be nice to have a starting point for optimization. Been reading about people having trouble transfecting this cell line with DNA.
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Lipofectamine® 2000 Reagent Protocol 2013
Lipofectamine® 2000 DNA Transfection Reagent Protocol
Transfect cells according to the following chart. Volumes are given on a per-well basis. Each reaction mix is sufficient for triplicate (96-well), duplicate (24-well), and
single well (6-well) transfections, and accounts for pipetting variations. Adjust the amounts of components according to your tissue culture format. For additional
information on scaling your transfection reaction, see page 1.
Timeline Steps Procedure Details
1
2
3
4
5
6
7
Day
1 Day 2–4 Day
0
5
Seed cells to be
70–90% confluent at
transfection
Component 96-well 24-well 6-well
Adherent cells 1–4 × 104 0.5–2 × 105 0.25–1 × 106
Dilute four amounts of
Lipofectamine® Reagent in
Opti-MEM® Medium
Opti-MEM® Medium 25 μL × 4 50 μL × 4 150 μL × 4
Lipofectamine® 2000
Reagent 1, 1.5, 2, 2.5 μL 2, 3, 4, 5 μL 6, 9, 12, 15 μL
Dilute DNA in Opti-MEM®
Medium
Opti-MEM® Medium 125 μL 250 μL 700 μL
DNA (0.5–5 μg/μL) 2.5 μg 5 μg 14 μg
Add diluted DNA to diluted
Lipofectamine® 2000 Reagent
(1:1 ratio)
Diluted DNA Total 25 μL 50 μL 150 μL
Diluted Lipofectamine®
2000 Reagent 25 μL 50 μL 150 μL
Incubate Incubate for 5 minutes at room temperature.
Add DNA-lipid complex to
cells
Component 96-well 24-well 6-well
DNA-lipid complex per
well 10 μL 50 μL 250 μL
Final DNA used per well 100 ng 500 ng 2500 ng
Final Lipofectamine® 2000
Reagent used per well 0.2–0.5 μL 1.0–2.5 μL 5.0–12.5 μL
Visualize/analyze
transfected cells Incubate cells for 1–3 days at 37°C. Then analyze transfected cells.
Hi,
BxPC3 is indeed a difficult cell line to transfect.
I optimized LF2000 (LF3000 was toxic) .The reaction is as follows for 100 mm plate:
tube 1:Opti-MEM- 500ul, Plasmid - 5ug
tube2: Opti-MEM -500ul,LF2000 - 5ul
Mix tube 1 and tube 2
Incubate at RT for 20 minutes.
Put it into the plate.
Incubate for 4-6 hours.
Change the media and keep the plate for 24-48h depending on your experiment.
P.S. LF2000 is toxic to BxPC3 cells beyond 6 hours.
Thank you, Nitika. I'm surprised to hear that LF2000 seems less toxic to these cells than LF3000. I will try your protocol for transfection of plasmid DNA. Cheers!
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