Especially developed for the transfection of mammalian cells with sirna or mirna

Transcription

1 METAFECTENE SI Especially developed for the transfection of mammalian cells with sirna or mirna For ordering information, MSDS, publications and application notes see Description Cat. No. Size METAFECTENE SI T ml METAFECTENE SI T ml METAFECTENE SI T ml Shipping: At room temperature (freeze on receipt!) Storage: METAFECTENE SI 15 C 10 SI buffer 15 C Stability: Best before: see label Use: Only for research purposes in vitro, not intended for human or animal diagnostic, therapeutic or other clinical uses. METAFECTENE SI is a composition of lipids especially developed for the transfection of mammalian cells with sirna or mirna. It generates excellent knockdown even with very small amounts of RNA, due to its new sicom technology. METAFECTENE SI is designed specifically for transfection with sirna and mirna, allowing for a protocol with a fixed RNA/reagent ratio which makes optimization a thing of the past. Furthermore, METAFECTENE SI is used with a reverse protocol, enabling two consecutive experiments to be performed per week (standard protocol with 48 h between addition of the lipoplex and evaluation). METAFECTENE SI Manual EN

2 Index 1. General Information Specifications Quality Control Explanatory Remarks... 3 Storage... 3 State of the cells... 3 Quality of the genetic material Working Instructions Prepare Reagents Prepare Cells Prepare Lipoplexes Transfection Evaluation Transfer to other formats Adjusting transfection parameters Amount of lipoplex Number of cells per well Miscellaneous Important Information Warranty Related Products METAFECTENE SI Manual EN

3 1. General Information 1.1 Specifications Application Formulation Sterility Assays Shelf life Storage Transfection of mammalian cells with sirna or mirna Composition of lipids in water Tested About 1000 (96-well) or about 250 (24-well) 1 year 15 C 1.2 Quality Control Standard transfection assay. Absence of bacterial and fungal contamination is verified using thioglycolate medium. 1.3 Explanatory Remarks Storage METAFECTENE SI is delivered non chilled and should be stored in a freezer at 15 C. The reagent should be thawed before each transfection to pass a freeze thaw cycle, which reconstitutes an optimal size distribution of the liposomes. The recommended storage conditions aim at this freeze thaw cycle and have nothing to do with the stability of the product. Shipping at room temperature does not affect its stability. Shipping at room temperature for several days does not affect the product's stability provided that it is afterwards stored again at 15 C. The reagent can be thawed for use and re-frozen any number of times. State of the cells Cells to be transfected should be well proliferating and healthy. Cells which have been in a quiescent state at total confluency for a while (before seeding) may not be transfected as efficiently as cells which are growing well (70 90% coverage of the growth area). It is therefore recommended to use only regularly passaged cells for transfection experiments. Microbial contamination, for example with mycoplasma or fungi, can drastically diminish transfection efficiencies. Biontex offers MycoSPY und MycoRAZOR for fast, reliable detection and efficient removal of mycoplasmal contaminations. Quality of the genetic material The RNA should be of the maximum purity if optimal knockdown results are desired. Note: RNA is sensitive to ubiquitous RNases. Appropriate precautions should therefore be taken! METAFECTENE SI Manual EN

4 2. Working Instructions 2.1 Prepare Reagents First of all dilute 1 aliquot of 10 SI buffer with 9 aliquots sterile water (suitable for cell culture) under sterile conditions to prepare 1 SI buffer. Before transfection bring the 1 SI buffer, METAFECTENE SI and the RNA stock solution to room temperature. Agitate all reagents gently before use. The following instructions refer to transfection in one well with a growth area of one square centimetre (one well of a 48-well plate). A table of specifications for other well formats is given in chapter 2.6. The specified amount of lipoplex (complex of RNA and respective volume of METAFECTENE SI) is suitable for most experiments. It can be adjusted according to the instructions in chapter 3.1, if necessary. 2.2 Prepare Cells Prepare 250 µl cell suspension in complete cell culture medium with a concentration of cells/ml. 2.3 Prepare Lipoplexes Pipet 30 µl of 1 SI buffer into a well. Then pipet 2 µl of METAFECTENE SI into the 1 SI buffer, followed by 30 pmol RNA, also pipetted into the SI buffer. Mix the contents of the well by gently pipetting up and down once. Incubate for 15 min at room temperature. 2.4 Transfection Add 250 µl of the prepared cell suspension to the well containing the lipoplex. Incubate the cells without further mixing under the usual conditions. 2.5 Evaluation Evaluation of the experiment (e.g. real time PCR or reporter gene assay) is done h after the addition of the lipoplex. The highest knockdown is often achieved after 48 h. The optimal point in time is determined by the properties of the cell type and the expression rate of the protein as well as by the half-life of the expressed protein, if applicable. 2.6 Transfer to other formats Format Growth area [cm²] 1 SI buffer [µl] METAFECTENE SI [µl] RNA [pmol] Cell suspension [µl] 96 well well well well well METAFECTENE SI Manual EN

5 3. Adjusting transfection parameters 3.1 Amount of lipoplex The amount of lipoplex added to the cells can be halved, e.g. to transfect very sensitive cells, or doubled to generate an even higher knockdown. To do so, halve or double the values given in the table in chapter 2.6 for both the volume of METAFECTENE SI and the amount of RNA. Do not change the volumes of the 1 SI buffer and the cell suspension. Always keep the ratio of METAFECTENE SI and RNA constant: 1 µl METAFECTENE SI for every 15 pmol of RNA 3.2 Number of cells per well If an increase in the number of cells per well is desired (and consequently a higher cell concentration when seeding the cells), the amount of lipoplex has to be increased proportionally. When doing so, keep the ratio of reagent to RNA at 1 µl METAFECTENE SI per 15 pmol RNA. METAFECTENE SI Manual EN

6 4. Miscellaneous 4.1 Important Information This reagent is developed and sold for research purposes and in-vitro use only. It is not intended for human therapeutic or diagnostic purposes. 4.2 Warranty Biontex guarantees the performance of this product until the date of expiry printed on the label when used in accordance with the information given in this manual. If you are not satisfied with the performance of the product please contact Biontex or one of its authorized distributors. 6 METAFECTENE SI Manual EN

7 5. Related Products Transfection Product Pack Size Cat. No. Product Pack Size Cat. No. METAFECTENE EASY + 2 T T METAFECTENE PRO 2 5 T T T METAFECTENE SI 2 T T METAFECTENE 2 5 T T T INSECTOGENE T T T T DOTAP T T T T Protein Purification Ni-NTA-Agarose 2 R R R R Ni-IDA-Agarose 2 R R R R Highflow Ni-IDA-Agarose 2 R R R R Pre-Packed L-Column Ni Pre-Packed XL-Column Ni Pre-Packed Ni-Cartridge 5 x Harz 5 x Harz Harz 5 x Harz P P P P Co-IDA-Agarose 2 R R R R Highflow Co-IDA-Agarose 2 R R R R Pre-Packed L-Column Co Pre-Packed XL-Column Co 5 x Harz 5 x Harz P P Pre-Packed GST-Cartridge Harz 5 x Harz P P GST-Agarose 2 R R R R Empty S-Columns Empty L-Columns Empty XL-Columns Empty Spin-Columns Empty Cartridges 50 x x 12 ml 50 x 3 50 x 800 µl 5 x P-50S P-50L P-50XL SP-50 C Proteofection PROTEOfectene 100 µl 250 µl 0. E E E E PROTEOfectene AB Reagents for mycoplasma-free cell culture 100 µl 250 µl 0. E E E E FBS MycoFREE (EU) FBS MycoFREE (US) FBS MycoFREE (AU) Trypsin MycoVIR (1 ) (1:250) 100 ml 500 ml 100 ml 500 ml 100 ml 500 ml M M M M M M ml M Trypsin MycoVIR (1 )+EDTA (1:250) 100 ml M Trypsin MycoVIR (10 )+ EDTA (1:250) 100 ml M MycoSPY Mycoplasmen-Detektionskit MycoRAZOR Mycoplasmen-Eliminierungskit 10 Assays 25 Assays 50 Assays M M M ml M Microfection Kits µ-transfection Kit VI 15 Slides K µ-proteofection Kit VI 15 Slides K µ-transfection Kit VI FluoR 15 Slides K µ-proteofection Kit VI AB 15 Slides K Visit the Biontex website (http://www.biontex.com) and select your home country to get specific contact and ordering information. METAFECTENE SI Manual EN

8 Biontex Laboratories GmbH Am Klopferspitz 19 im IZB Planegg/Martinsried Germany Phone: +49 (0) Fax: +49 (0) Fax 2: +49 (0) Internet: 8 METAFECTENE SI Manual EN