Magnetofectamine™

Reverse InstructionManual

The twomostpowerful transfectiontechnologies in onekit!

Magnetofectamine™ istheassociationofthefamous Lipofectamine™ 2000 transfection reagentfrom Invitrogen™, with theunique Magnetofection™ based reagent CombiMag, fromOZBiosciences™

Listof Magnetofectamine™ kits. Catalog Numberoftransfection Description Volume(µL) Number per µgofDNA MTX0750 Magnetofectamine™ Kit 1 250µL+ 750µL 250-375 MTX1000 Magnetofectamine™ StartingKit 2 250µL+750µL 250-375 1 Contains 1vial ofeachreagent (250µL of CombiMag + 750µLof LipofectamineTM 2000) 2 Contains 1vial ofeachreagent andaMagneticplate (250µLofCombiMag+ 750µLof LipofectamineTM 2000 +SuperMagneticplate 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 hesitatetocontact ourdedicatedtechnicalsupport ([email protected]).

OZBIOSCIENCES ParcScientifique deLuminy 163 avenue deLuminy, Zoneentreprise,case 922 13288 MarseilleCedex 9, France Tel:+33 (0) 486 948 516 Fax: +33(0)486 948 515 E-mail: [email protected] WebSite: www.ozbiosciences.com

Distributed by

Lifescience&diagnosticsolutions

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.

OZBiosciences/Protocol Magnetofectamine – Suspensioncells/www.ozbiosciences.com/ - 1 -

MEDIBENA LifeScience&DiagnosticSolutions,Zeillergasse38,1170Wien, Austria TEL:+43-1-9906-497,FAX:+43-1-2533-0339-577, [email protected]Registerofcompanies:HandelsgerichtWienÖsterreichFN:330781v,VAT: ATU65121744 ReverseTransfection 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 DNA and siRNA reverse transfection into mammalian cells using Magnetofectamine™. Formore informationon culture,transfection volumesand optimization please referto thegeneralprotocol.

Importantconsiderations beforebeginningtransfection: x Use low-passage cells, and make sure that cells are healthy and greater than 90% viable before transfection. x Transfectcells at80-90%confluence. x Do not add antibiotics to the medium during transfection as this reduces transfection efficiency and causes celldeath. x Alwaysuse avolume of reagent thatisatleasttwice thequantity of nucleicacid (2µL per µgof DNA). x Forco-transfectionofseveralDNA, use total DNAquantityfor complexespreparation.

1) Reagents preparation

Pleaserefertothegeneral protocol fortherecommendedquantitiesof DNA,siRNAandreagents.

Foreachtransfectionsample,prepareDNA(siRNA)/ MagnetofectamineTM complexesas follow:

a. Nucleic acids. Dilute the DNA or siRNA molecules in an appropriate amount of Opti-MEM® I Medium withoutserum. 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 1µL CombiMag per µg DNA or 1 µLfor 40 pmolofsiRNA.Addthe CombiMagreagent directlyintoatube(donotdilute with anyculturemedium).

Note: For CombiMag volumes less than 1µL, first dilute the reagent with deionized water only and use thevolumerequired foryour DNAor siRNAdose. DiscardthedilutedCombiMagafteruse.

Note: Proceedtostep 2 within10minutes

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 gentlyandincubate for 20minutesatroomtemperature.

Note: Proceedtostep 4 within 30 minutes

3) Transfection preparation. a. Place theculture plate tobe transfectedontothemagneticplate. b. Addthe complexestothe wellsandincubate 5 min ontothemagnetic plate.

4) Transfection. c. Keeptheculture plateonto the magneticplate d. Carefullyadd cellsuspension ineachwellcontainingthecomplexes. 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 magneticplate.

5) Assay. Incubate the cells at 37°C in a CO2 incubator for 24 to 96 hours until evaluation of the expression or genesilencing.

Note: Forsomecells, the medium canbe changedafter 4-6 hours of incubation withfreshmedium.

OZBiosciences/Protocol Magnetofectamine – Suspensioncells/www.ozbiosciences.com/ - 2 -