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Lipofectamine® 2000 Reagent

Cat. nos. 11668-030 11668-027 11668-019 11668-500 Size: 0.3 mL 0.75 mL 1.5 mL 15 mL

MAN0000995

Store at 4°C (do not freeze)

Publication Part No. 11668.2k.pps

Rev. Date 12 March 2012

Description

· Lipofectamine®2000TransfectionReagentisaproprietaryformulation fortransfectingnucleicacids(DNAandRNA)intoawiderangeof eukaryoticcells. Nucleicacid-Lipofectamine®2000complexescanbeaddeddirectlyto cellsinculturemedium,inthepresenceorabsenceofserum. Itisnotnecessarytoremovecomplexesorchange/addmediumafter transfection.

· ·

Important Guidelines for Transfection

· · UseOpti-MEM®IReducedSerumMedium(Cat.no.31985-062)to diluteLipofectamine®2000TransfectionReagentandnucleicacids. TheamountofLipofectamine®2000Reagentrequiredforsuccessful transfectionvariesdependingonthecelltypeandpassagenumber. Startanynewtransfectionbytesingtherecommendedfour concentrationsofLipofectamine®2000Reagenttodeterminean optimumamount. Preparehigh-qualityplasmidDNAat0.5­5µg/µL in deionized water or TE buffer. A GFP (green fluorescent protein) plasmid can be used to determine transfection efficiency. Foradditionalinformation,andprotocolsontransfectingsuspension cellsrefertothemanualatwww.lifetechnologies.com/manuals.

·

·

For research use only. Not for human or animal therapeutic or diagnostic use.

Cells Day 0

Seed cells so they are 70­90% confluent at the time of transfection.

Plasmid Transfection

Transfectcellsaccordingtothefollowingtable.Volumesaregivenonaper wellbasis.Varytheamountofcomponentsaccordingtoyourtissueculture format.Foradditionalinformationonscalingyourtransfectionreaction,see page4. Component

Adherent cells Opti-MEM® Medium Lipofectamine 2000 Reagent

®

Lipofectamine® 2000 Reagent

Dilute 4 amounts of Lipofectamine® 2000 Reagent in Opti-MEM® Medium.

96-well

1­4 x 104 50 µL x 4 2, 3, 4, 5 µL 250 µL 5 µg 50 µL 50 µL

24-well

0.5­2 x 105 50 µL x 4 2, 3, 4, 5 µL 250 µL 5 µg 50 µL 50 µL

6-well

0.25­1 x 106 150 µL x 4 6, 9, 12, 15 µL 700 µL 14 µg 50 µL 50 µL

DNA

Dilute DNA in Opti-MEM® Medium.

Opti-MEM® Medium

Day 1

DNA (0.5­5 µg/µL) Diluted DNA Diluted Lipofectamine® 2000 Reagent

Add diluted DNA to each tube of diluted Lipofectamine® 2000 Reagent (1:1 ratio).

Incubate for 5 minutes at room temperature DNA-reagent complex/well 10 µL 50 µL 250 µL

5

Incubate.

Incubate cells for 1­3 days at 37°C

Add DNA-reagent complex to cells.

ThefollowingtableshowstheamountsofDNAandLipofectamine®2000 Reagentusedineachtransfectionreaction. Amount 96-well

100 ng

®

24-well

500 ng 1­2.5 µL

6-well

2500 ng 5­12.5 µL

Day 2­4

Visualize/analyze transfected cells.

DNA/well Lipofectamine 2000 Reagent/ well

0.2­0.5 µL

Scaling Up or Down Transfections

Usethemultiplicationfactorinthefollowingtabletoscalethevolumesfor yourtransfectionexperiment.Thefactorisbasedontherelativesurfacearea ofasinglewellfroma24-wellplate.

Culture Vessel 96-well plate 48-well plate 24-well plate 12-well plate Multiplication Factor 0.2X 0.5X 1X 2X Culture Vessel 6-well plate 60 mm dish 100 mm dish T75 flask Multiplication Factor 5X 11X 29X 39X

Co-Transfection of Plasmid DNA and siRNA

PlasmidDNAandsiRNAcanbetransfectedatthesametimeusing Lipofectamine®2000Reagentbyadding40pmol(~0.6µg)ofsiRNAper 1µg of DNA.

mRNA Transfection

mRNAcanbetransfectedina24-wellplatebyusingLipofectamine®2000 Reagentbyadding0.5­1µgofmRNAperwell.

LIMITED USE LABEL LICENSE: RESEARCH USE ONLY

The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser's activities for a fee or other form of consideration. For information on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

Limited Product Warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at www.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support.

LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. ©2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

For support visit www.lifetechnologies.com/support www.lifetechnologies.com

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