STRATAGENE to Transfection
Contents
1. e ooo o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 2 o o o 0 Table 1 Selection Guide for Transfection Reagents RECOMMENDED TRANSFECTION FEATURES INCUBATION APPLICATION METHOD TIME Mammalian Transfection Kit DNA transfection CaP04 DEAE Works for many cell types 12 24 hrs MBS Mammalian Transfection Kit DNA transfection Modified CaP04 Works with low DNA concentration SNS Recommended for CHO and HEK293 cells LipoTaxi Transfection Reagent DNA transfection Liposome based Works for many cell types 4 6hrs Requires small amounts of DNA Gentle on cells Room temperature storage GeneJammer Transfection Reagent DNA transfection Polyamine based Good for hard to transfect 3 hrs e g primary cells T Works for many cell types Effective in presence of serum no media changes required T Less cytotoxic than lipids GeneEraser siRNA Transfection Reagent siRNA transfection Proprietary formulation Optimized for siRNA 20572 firs 1 transfection into many cell types Knocks down target gene even at low siRNA concentration Low cell cytotoxicity Works in presence of serum BioTrek Protein Delivery Reagent Protein peptide Lipid form2. the closed tube real time detection format minimizes the potential for cross contamination with PCR amplicon We include a passive reference dye in the kit to allow you the flexibility of running the assay on most real time QPCR platforms The kit also contains DNA purification reagents for removal of potential PCR inhibitors in cell culture supernatants and cell pellets The MycoSensor QPCR assay kit provides a fast convenient method for the routine testing of cell cultures for mycoplasma and acholeplasma contamination The kit delivers consistent results and includes controls designed to eliminate false negatives and false positives We ve optimized the MycoSensor QPCR assay kit for maximum performance on multiple real time QPCR platforms including our Mx3000P and Mx4000 Quantitative PCR instruments the Cephied Smart Cycler and the ABI PRISM 7700 mycoplasma 82 C im Fluorescence dRn Temperature C 54 56 58 60 62 64 66 68 70 72 74 16 18 80 82 84 86 88 90 92 94 se e o gt o o o o o o o o o o oo oo o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o oe Ordering Information Description Catalog No Size Price Mycoplasma Detection Kits MYCOPLASMA PLUS PCR PRIMER SET 1 kit 100 rxn 302008
3. 335 MYCOSENSOR PCR ASSAY KIT 1 kit 100 rxn 302108 335 MYCOSENSOR QPCR ASSAY KIT 1 kit 100 rxn 302106 395 Transfection Reagents amp Kits GENEJAMMER TRANSFECTION REAGENT 0 3 ml 204132 102 1 0 ml 204130 202 4 x 1 0 ml 204131 728 LIPOTAXI TRANSFECTION REAGENT 3x1ml 204110 211 MAMMALIAN TRANSFECTION KITS CAPO4 AND DEAE 1 kit 30 transfections each 200285 237 CAPO4 1 kit 30 transfections 200385 113 DEAE 1 kit 30 transfections 200386 113 MBS MAMMALIAN TRANSFECTION KIT 1 kit 200388 330 Protein Delivery BIOTREK PROTEIN DELIVERY REAGENT 1 kit 24 rxn 204140 270 siRNA Transfection GENEERASER SIRNA TRANSFECTION REAGENT 0 4 ml 204152 177 1 0 ml 204150 358 Viral Delivery VIRAPACK TRANSFECTION KIT 1 kit 200488 190 Other Related Products BETA GALACTOSIDASE ASSAY KIT 1 kit 100 rxn 200383 149 BETA GALACTOSIDASE ASSAY KIT HIGH SENSITIVITY 1 kit 100 rxn 200710 180 BETA GALACTOSIDASE ASSAY KIT IN SITU 1 kit 200384 154 G418 SULFATE 100 mg 200397 27 500 mg 200398 52 1 mg 200399 93 10 ml 200049 62 HYGROMYCIN B 1 MU 1 x 106 U 200715 129 LUCIFERASE ASSAY KIT 1 kit 100 rxn 219020 90 QUANTOS CELL PROLIFERATION ASSAY KIT 1 kit 302011 221 LEGAL INFORMATION 1 US Patent No 5 330 904 2 BioTrek Protein Delivery Reagent is manufactured for Stratagene by Gene Therapy Systems Inc 3 GeneEraser siRNA Transfection Reagent is manufactured for Stratagene by Mirus 4 Patent pending Purchase of this PCR related product does no
4. 7400 7400 Stratagene Japan K K Order 3 5821 8077 Technical Services 3 5821 8076 Distributors 5 For a list of worldwide distributors please visit www stratagene com Stratagene an Agilent Technologies company 2008 Printed in USA April 2008 BR61 5989 8234EN STRATAGENE An Agilent Technologies Company 11011 North Torrey Pines Road La Jolla CA 92037 USA PRORT SID U S POSTAGE PAID San Diego CA Permit No 1 BR61
5. GHT MOST COMMON SPECIES OF MYCOPLASMA AND ACHOLEPLASMA 2 MM arginini 4 M hominis 6 M orale 8 M salivarium Lanes 1 2 3 4 5 6 www stratagene com Fluorescence dRn TRANSFECTION gt MYCOPLASMA DETECTION Mycoplasma Detection by Real Time PCR Our new MycoSensor QPCR Assay Kit detects mycoplasma infection of cell cultures by real time quantitative PCR using SYBR Green dye detection in less than 2 hours The conven ient master mix format provides all the reagents needed for amplification and fluorescence detection and the kit detects the eight most common cell culture contaminating species of mycoplasma Table 4 Customers can detect as few as 10 copies of contaminating genomic DNA from 100 ul of cell culture supernatant or test for weak mycoplasma infection in extracts made from cell pellets Figure 8 The kit includes two positive control templates to validate the detection of successful PCR amplification of mycoplasma template and confirm the SYBR Green dissociation profile for your test samples If your test cell line is infected the SYBR Green dissociation profile results in a melting peak at 82 C As seen by a melting peak around 85 C the amplification control confirms the absence of PCR inhibitors thus reducing false negative results Figure 9 To minimize false positives 100 000 10 000 1 000 100 2 4 6 amp 6 8 10 12 14 16 18 20 2 24 25 28 30 32 34 35 38 40 Temperature C
6. PROTEIN FUNCTION amp ANALYSIS Transfection How do you study and control gene expression How do you detect mycoplasma contamination How do you ensure the success of your RNA or protein transfection o 8 6 8 5 5 6 oc A o s 8 HK we a of 8 46 c o MN o 7 7 Ae o DIE co FH OT s o Ness si O AA PAA AA AA AA AO sits STRATAGENE An Agilent Technologies Company Introduction to Transfection Transfection is the delivery of DNA RNA proteins and macromolecules into eukaryotic cells Goals for transfection include the study of gene regulation as well as protein expression and function The success of transfection depends on transfection efficiency low cytotoxicity and reproducibility To ensure high efficiency of your transfection you need reliable transfection technology and reagents to optimize your cell culture conditions We provide you with a variety of reagents for your preferred transfection method and mycoplasma detection kits to ensure early detection of cell culture contamination For more information on our transfection products please visit www stratagene com transfection CONTENTS Transfection Mycoplasma Transfection Selection Guide 6 Mycoplasma Selection Guide Transient and Stable Transfections 7 8 Mycoplasma Detection Special Transfection Needs 9 Ordering Information se
7. at room temperature easy to transfect COS 7 cell line 100 n 80 pa c o D 5 60 c o 5 v 5 40 o S Ee 20 GeneJammer Lipofectamine FuGENE6 GenePORTER 2 SuperFect Lipofectamine 2000 Plus difficult to transfect HUVEC primary cell line 25 4 20 c Pa o D a 15 c o 5 2 a 10 S Ee GeneJammer Lipofectamine 8FUGENE 6 GenePORTER 2 SuperFect Lipofectamine 2000 Plus Polyamines Our GeneJammer Transfection Reagent provides the high transfection efficiencies that you need even in the most problematic conditions such as in the presence of serum or in transfection of primary cells Figure 3 Furthermore this easy to use reagent results in significantly reduced levels of cell damage as compared to the traditional liposomal method Figure 4 Cytotoxicity Comparison In HeLa Cells 100 viable cells GeneJammer Lipofectamine FuGENE 6 GenePORTER 2 SuperFect Lipofectamine 2000 Plus www stratagene com TRANSFECTION gt SPECIAL TRANSFECTION NEEDS Special Transfection Needs We also offer a variety of specialty transfection reagents and kits to meet your experimental needs such as transfecting RNA proteins or for virus packaging Protein Delivery Our BioTrek Protein Delivery Reagent allows you to directly translocate your protein into a living cell Conveniently packaged in single use vials this provides an easy method t
8. e transfected Figure 1 Both are relatively simple techniques with DEAE dextran better suited for transient transfections and the calcium phosphate method for Stable transfections COLONIES PER PLATE Standard CaPO M Lipofectin Is Mammalian Transfection Kit CaPO i 0 200 400 600 800 1000 1200 l l l l l To increase transfection efficiency over these standard methods we utilize a specially modified bovine serum in our MBS Mammalian Transfection Kit for use during transfection This results in significantly higher efficiencies as well as reduced assay time Figure 2 This kit exhibits superior performance with CHO Chinese Hamster Ovary and HEK293 Human Embryonic Kidney cells B GALACTOSIDASE UNITS MG CELL PROTEIN TRANSFECTION TIME Okayama amp Chen 3 hours Lipofection 6 hours ir EJ meu ASAN Eb MBS Mammalian Transfection Kit Sa En 3 hours S Experiment 1 CHO cells Experiment 2 CHO cells 1 10 100 1 000 10 000 100 000 l l l Lipofection Compared to the DEAE dextran and calcium phosphate methods liposomes often offer higher transfection efficiency and better reproducibility Using our LipoTAXI Transfection Reagent results in stable and transient transfections with low cytotoxicity and consistent high transfection efficiencies a 10 fold increase over calcium phosphate DEAE mediated transfection in many cell lines Furthermore our LipoTAXI reagent can be conveniently stored
9. ion of carry over PCR product Detects as few as 10 copies of mycoplasma genomic DNA depending on species MycoSensor QPCR Assay Kit Real Time QPCR Quick results 2 hours Detection Convenient master mix format Detects as few as 10 copies of mycoplasma genomic DNA depending on species Includes positive and internal amplification controls Includes PCR purification kit Tested on multiple real time QPCR platforms Mycoplasma Detection Traditional mycoplasma detection methods such as culture Our next generation PCR amplification kit the MycoSensor testing staining and immunofluorescence detection are PCR Assay Kit features enhanced primer design to improve very time consuming not sensitive and difficult to interpret detection of the eight most common contaminating species PCR amplification provides a quick and easy method for Table 4 and to prevent the detection of E coli DNA which mycoplasma detection often contaminates recombinant Tag DNA polymerase preparations The MycoSensor kit features shorter run time The Mycoplasma Plus PCR Primer Set provides you reliable 50 faster and detects as few as 10 copies of Mycoplasma PCR detection and speciation of mycoplasma This kit provides genomic DNA depending on species Figure 7 an internal control to confirm PCR amplification was successful and a noninfectious mycoplasma genomic DNA template that serves as a positive control EI
10. o effectively deliver proteins peptides and other macromolecules directly into a broad range of cell types at efficiencies of 50 95 in as little as 3 4 hours Figure 5 This enables you to rapidly and accurately study cellular and protein function Figure 5 PROTEIN TRANSFECTION OF BETA GALACTOSIDASE WITH BIOTREK REAGENT BioTrek reagent mediated intracellular delivery of beta galactosidase in NIH 3T3 cells A After 4 hours cells were fixed stained for activity with the X gal reagent then compared to standard transfection B siRNA Transfection High Titer Virus Production RNA interference RNAi is a powerful method for inhibiting The transfection efficiency of the packaging cell line in any the expression of a particular gene RNAi experiments require viral delivery experiment is critical Our ViraPack Transfection a transfection reagent that is specially optimized for siRNA Kit transfects the HEK293 packaging cell line with exceptional delivery into mammalian cells efficiency so you can Increase the number of infectious virus particles produced per transfection Our GeneEraser siRNA Transfection Reagent enables the use of very low levels of siRNA to knock out stable or transient gene expression in a wide variety of cell lines and has a high trans fection efficiency Figure 6 Provided in a ready to use format the GeneEraser reagent and your siRNA can be added directly to cells in media even in the presence of
11. serum s o e o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o siRNA CONCENTRATION nM an a __ AAA 80 60 40 I 0 2 10 20 30 10 20 30 10 20 30 GeneEraser Reagent A Reagent B Luciferase Expression www stratagene com Mycoplasma Detection Present in 5 35 of cell cultures mycoplasma contamination can alter growth characteristics enzyme patterns cell membrane composition chromosomal structure and transfection efficiency These lead to unreliable experimental results as well as loss of time and precious cell lines Therefore routine screening for mycoplasma contamination is essential Unlike bacteria and fungi mycoplasma contamination cannot be detected by visual inspection Their small size allows them to pass through most sterilizat sion filters and they are resistant to common antibiotics Table 3 Mycoplasma Selection Guide METHOD FEATURES RECOMMENDED APPLICATION Mycoplasma Plus PCR Primer Set Includes positive and Detection speciation internal controls MycoSensor PCR Assay Kit L Primer design prevents detection of Detection E coli DNA that often contaminates recombinant Tag DNA polymerase preparations Short cycle time 50 faster Protocol ensures decontaminat
12. t convey any rights under the PCR patents owned by Hoffmann La Roche A license to use the PCR process accompanies the purchase of certain reagents from Stratagene when used in conjunction with an Authorized Thermal Cycler 5 Patent pending Purchase of these products is accompanied by a license to use them in the Polymerase Chain Reaction PCR process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up front license fee either by payment to Applied Biosystems or as purchased i e an authorized thermal cycler SYBR is a registered trademark of Molecular Probes FUGENE 6 is a registered trademark of Fugent LLC USA GenePORTER 2 is a trademark of Gene Therapy Systems Inc Lipofectamine Lipofectamine Plus and Lipofectamine 2000 are trademarks of Invitrogen Corp SuperFect is a registered trademark of Qiagen Inc SYBR is a registered trademark of Molecular Probes se e o o o o o o o o o o o o o o o o e e i www stratagene com Stratagene USA and Canada Order 800 424 5444 x3 Technical Services 800 894 1304 x2 Stratagene Europe Order 00800 7000 7000 Technical Services 00800
13. ulation Works for many cell types A his macromolecule delivery 1 Conveniently packaged in single use vials Includes control proteins ViraPack Transfection Kit Viral packaging CaPO4 Optimized for viral packaging cell lines SIS s e o o gt o o gt o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o l l l l M l T T l l l TH l l l H Tl l I I l IM T I I I IM I I I I i I I I I I T I I I T T I I I I T l o o ooo 6 8200 www stratagene com TRANSFECTION gt TRANSIENT AND STABLE TRANSFECTIONS Transient and Stable Transfections Classical transfection techniques include the DEAE dextran method calcium phosphate method and liposome mediated transfection lipofection Your choice of transfection technology will influence your results We offer a variety of transfection reagents and kits to suit your specific needs DEAE dextran and Calcium Phosphate Methods We offer several mammalian transfection kits based on the original methods used to introduce DNA into cultured mammalian cells DEAE dextran and calcium phosphate Our original Mammalian Transfection Kit utilizes the modified calcium phosphate method which yields up to 100 fold efficiency over traditional methods depending on the cell line to b
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STRATAGENE to Transfection