Magnetofectamine™
Reverse Transfection Instruction Manual
The two most powerful transfection technologies in one kit!
Magnetofectamine™ is the association of the famous Lipofectamine™ 2000 transfection reagent from Invitrogen™, with the unique Magnetofection™ based reagent CombiMag, from OZ Biosciences™
List of Magnetofectamine™ kits. Catalog Number of transfection Description Volume (µL) Number per µg of DNA MTX0750 Magnetofectamine™ Kit 1 250µL + 750µL 250-375 MTX1000 Magnetofectamine™ Starting Kit 2 250µL + 750µL 250-375 1 Contains 1 vial of each reagent (250µL of CombiMag + 750µL of LipofectamineTM 2000) 2 Contains 1 vial of each reagent and a Magnetic plate (250µL of CombiMag + 750µL of LipofectamineTM 2000 + Super Magnetic plate MF10000)
Use the content of the table above to determine the appropriate catalog number for your needs. You can order these products by contacting us ([email protected]). For all other supplementary information, do not hesitate to contact our dedicated technical support ([email protected]).
OZ BIOSCIENCES Parc Scientifique de Luminy 163 avenue de Luminy, Zone entreprise, case 922 13288 Marseille Cedex 9, France Tel: +33 (0) 486 948 516 Fax: +33 (0) 486 948 515 E-mail: [email protected] Web Site: www.ozbiosciences.com
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Lipofectamine™ and Invitrogen™ are Trademarks owned by Life Technologies Corporation. Lipofectamine™ 2000 is manufactured by Life Technologies Corporation for OZ Biosciences and provided under license from Life Technologies Corporation.
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MEDIBENA Life Science & Diagnostic Solutions, Zeillergasse 38, 1170 Wien, Austria TEL: +43-1-9906-497, FAX: +43-1-2533-0339-577, [email protected] Register of companies: Handelsgericht Wien Österreich FN: 330781v, VAT: ATU65121744 Reverse Transfection Protocol
Reverse transfection combines DNA or RNA, transfection reagent, and cells in an altered sequence compared to traditional Lipofectamine™ 2000 or Magnetofectamine™ transfection protocols. The complexes are prepared inside the wells, after which cells and medium are added. This reference protocol provides general guidelines and procedure for plasmid DNA and siRNA reverse transfection into mammalian cells using Magnetofectamine™. For more information on cell culture, transfection volumes and optimization please refer to the general protocol.
Important considerations before beginning transfection: x Use low-passage cells, and make sure that cells are healthy and greater than 90% viable before transfection. x Transfect cells at 80-90% confluence. x Do not add antibiotics to the medium during transfection as this reduces transfection efficiency and causes cell death. x Always use a volume of reagent that is at least twice the quantity of nucleic acid (2µL per µg of DNA). x For co-transfection of several plasmids DNA, use total DNA quantity for complexes preparation.
1) Reagents preparation
Please refer to the general protocol for the recommended quantities of DNA, siRNA and reagents.
For each transfection sample, prepare DNA (siRNA) / MagnetofectamineTM complexes as follow:
a. Nucleic acids. Dilute the DNA or siRNA molecules in an appropriate amount of Opti-MEM® I Medium without serum. b. Lipofectamine™ 2000. Mix Lipofectamine™ 2000 gently before use and then dilute the appropriate amount in Opti-MEM® I Medium without serum. Mix gently and incubate 5 minutes at room temperature. c. CombiMag reagent. Vortex the reagent before each use. Use 1 µL CombiMag per µg DNA or 1 µL for 40 pmol of siRNA. Add the CombiMag reagent directly into a tube (do not dilute with any culture medium).
Note: For CombiMag volumes less than 1µL, first dilute the reagent with deionized water only and use the volume required for your DNA or siRNA dose. Discard the diluted CombiMag after use.
Note: Proceed to step 2 within 10 minutes
2) Complexes formation. Combine the nucleic acids solution with the Lipofectamine™ 2000 solution. Mix gently and add the nucleic acids/Lipofectamine™ 2000 mixture immediately to the CombiMag Reagent. Mix gently and incubate for 20 minutes at room temperature.
Note: Proceed to step 4 within 30 minutes
3) Transfection preparation. a. Place the culture plate to be transfected onto the magnetic plate. b. Add the complexes to the wells and incubate 5 min onto the magnetic plate.
4) Transfection. c. Keep the culture plate onto the magnetic plate d. Carefully add cell suspension in each well containing the complexes. e. Incubate the cells for 30 minutes on the magnetic plate at room temperature or at 37°C in a CO2 incubator. After this incubation period, remove the magnetic plate.
5) Assay. Incubate the cells at 37°C in a CO2 incubator for 24 to 96 hours until evaluation of the gene expression or gene silencing.
Note: For some cells, the medium can be changed after 4-6 hours of incubation with fresh medium.
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