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PerFectin Transfection Reagent Reactions

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					                                                                                                         A division of Gene Therapy Systems, Inc.


                          PerFectin™ Transfection Reagent (200 Reactions)
               PRODUCT SUMMARY
                   Cat. No:          T303015

                   Description:      PerFectin Transfection Reagent is a cationic lipid specifically formulated and optimized to
                                     work with CHO, COS, 293, NIH-3T3, HeLa, and Jurkat cells. One vial of PerFectin
                                     reagent (1.5 ml) is sufficient for 200 transfections using 2 µg of DNA per well.

                   Components: Dried PerFectin lipids film (one vial)
                               Hydration buffer (one vial, 1.6 ml)
                               DNA diluent (one vial, 10.5 ml)

                   Storage:          Store components at 4oC.

                   Stability:        Dried PerFectin reagent is stable for at least 2 years at 4oC.
                                     Hydrated PerFectin reagent is stable for at least 1 year at 4oC.
                                     DNA diluent is stable for at least 2 years at 4oC.


               INTRODUCTION
                   PerFectin Transfection Reagent is a unique formulation of the neutral lipid dioleoyl
                   phosphatidylethanolamine (DOPE) and a proprietary cationic lipid derived from Direct Hydrophilic
                   Conjugation (DHC) technology. PerFectin reagent has been specifically optimized for transfection and
                   gene expression in CHO, COS, 293, NIH-3T3, HeLa, and Jurkat cells. Each PerFectin reagent kit includes
                   a special DNA diluent buffer that increases transfection efficiency in the presence of serum. PerFectin
                   reagent is suitable for both in vitro and in vivo transfection studies. Perfectin reagent offers:

                   •   Superior transfection efficiency
                   •   Cost effectiveness
                   •   Compatibility with Serum
                   •   Extended shelf life


                                          Table 1: Cell Types in Which PerFectin is Optimized
                                          PerFectin reagent was tested and the formulation and
                                          protocols were optimized in the following cell types.

                                                           293              HeLa-S3
                                                         CHO-K1              Jurkat
                                                          COS-7             NIH-3T3




Version MV050506                               10190 Telesis Court, San Diego, CA 92121                                Page 1 of 1
                                Phone: (858) 457-1919 US Toll-Free: 888-428-0558 Fax: (858) 623-9494
                                                   Website: http:/www.genlantis.com
               DIRECTIONS
               •    Hydrate PerFectin lipid film at room temperature with 1.5 ml of the hydration buffer. Vortex for 10
                    seconds at top speed before use. Store the hydrated reagent at 4oC and vortex briefly before each use.
               •    Use the DNA diluent to prepare the DNA solution. Use 25 µl of diluent for 1 µg DNA.
               •    Use 3.5 µl of PerFectin reagent with 1 µg of DNA.

               EXAMPLE PROTOCOLS
               Transfection of 293, CHO-K1, COS-7, HeLa-S3, and NIH-3T3 cells
               1.   The day before transfection, plate the cells so that they will be 50% to 70% confluent on the day of
                    transfection

               2.   Dilute the hydrated PerFectin reagent with serum-free medium.
                    • Refer to Table 2 for the appropriate volume of serum-free medium.

               3.   Dilute the DNA with the DNA diluent, mix well by pipetting several times and incubate 5 minutes at
                    room temperature.
                    • Refer to Table 2 for the appropriate volume of DNA diluent.
                    • Avoid vortexing the DNA diluent solution.

               4.   Add the DNA solution to the diluted PerFectin reagent. Incubate at room temperature for 5 minutes to
                    form PerFectin /DNA complexes (lipoplexes).
                    • Do not incubate longer than 30 minutes.

               5.   Add the PerFectin /DNA complexes directly to the cells that are in serum-containing culture medium.
                    Incubate at 37oC in a humidified atmosphere containing 5% CO2.

                    •    For some cells (HeLa-S3), higher transfection efficiencies can be achieved when the initial 4-hour
                         incubation is done in serum-free medium. Use the following four steps:
                         (i) Add serum-free medium to the complexes to make up the transfection volume (refer to table 3).
                         (ii) Aspirate the culture medium from cells.
                         (iii) Add the complexes in serum-free medium to the cells. Incubate 4 hours at 37oC and 5% CO2.
                         (iv) Add one volume of medium containing 20% serum, then proceed to step 6.

               6.   24 hours post transfection, add fresh growth media as needed.
                    • For some cell types, the old media can be replaced with fresh media at this step.

               7.   Assay the reporter gene 24 to72 hours following transfection.
                    • Depending on the cell type and promoter activity, the same protocol can be used to produce stably
                        transfected cells: 48 to 72 hours post transfection, put the cells in fresh medium containing the
                        appropriate selection antibiotic. It is important to wait at least 48 hours before exposing the
                        transfected cells to the selection media.

               Table 2                                                          Table 3
                                                                               Transfection Volumes and DNA Amount for
                                                   a
              Volumes of Transfection Reagents                                 Various Culture Dishes a
                     DNA diluent     PerFectin           PerFectin                                        Transfection
              DNA                                        diluent (Serum            Tissue        DNA        Volume
               (µg)       (µl)          (µl)           free medium) (µl)        Culture Dish      (µg)        (ml)
                0.5       12.5          1.75                   10.75              96-well       0.1-0.5       0.1
                 1         25            3.5                   21.5               24-well        0.5-2        0.25
                 2         50            7                     43                  6-well          2-6         1
                 4        100            14                    86                 60 mm            6-8        2.5
                 8        200            28                    172                100 mm          8-12         5



Version MV050506                            10190 Telesis Court, San Diego, CA 92121                            Page 2 of 2
                             Phone: (858) 457-1919 US Toll-Free: 888-428-0558 Fax: (858) 623-9494
                                                Website: http:/www.genlantis.com
               Transfection of Jurkat cells

               The protocol for Jurkat cells is similar to the protocols described above, with the following exceptions.

               1.     The day before transfection split the cells so they are in good condition on the day of transfection.

               2.     Prepare the mixture of PerFectin /DNA complexes as above. While the PerFectin /DNA complexes are
                      incubating, spin down the cells, resuspend them at 1 x 106 or 2 x 106cells/ml in medium, and transfer the
                      appropriate volume to the dish (Table 4). Serum-free medium is preferred to resuspend the cells.

               3.     Add the complex directly to the cells, and mix well by gently pipetting 2 to 3 times. This step is important
                      because some Jurkat cells have a tendency to clump, and the reagent does not easily access cells in the
                      center of clumps. Gentle pipetting of cells disrupts these clumps and produces a true single-cell
                      suspension, which will increase transfection efficiency.

               4.     Incubate the cells at 37oC in a humidified atmosphere containing 5% CO2.

               5.     4 hours post transfection; add one volume of medium containing 20% serum to the cells.

               6.     24 hours post transfection; add fresh growth media as needed.

               7.     Assay the reporter gene 24 to72 hours following transfection.
                       • If low expression level is experienced, use serum-free medium instead of DNA diluent to dilute the
                           DNA. Also try low DNA amount at ~ 7 µl PerFectin per µg DNA ratio.
                       • Depending on the cell type and promoter activity, the same protocol can be used to produce stably
                           transfected cells: 48 to 72 hours post transfection, put the cells in fresh medium containing the
                           appropriate selection antibiotic. It is important to wait at least 48 hours before exposing the
                           transfected cells to the selection media.

                                          Table 4
                                                                                                    a
                                          Suggested Conditions for Transfecting Jurkat Cells
                                            Tissue                              Transfection
                                          Culture Dish   Number of Cells          Volume
                                                                    5
                                            96-well          1 x 10                0.1 ml
                                                                            6
                                              24 well            0.5 x 10                0.25 ml
                                                                        6
                                              6-well              2 x 10                  1 ml
                                                                        6
                                              60 mm               5 x 10                 2.5 ml
                                                                        7
                                             100 mm               1 x 10                  5 ml



               Notes
               a
                   To obtain maximum efficiency in your particular cell lines, additional optimization may be needed. The two
                   critical variables are the ratio of PerFectin reagent to DNA and the quantity of DNA. First maintain a fixed
                   ratio of PerFectin reagent to DNA, and then vary the DNA quantity over the suggested range. If necessary,
                   optimize the ratio of PerFectin reagent to DNA by using 2 to 6 µl of reagent for each 1 µg of DNA. Use a low
                   DNA quantity to optimize this ratio. Following this process, cell number can also be optimized.




Version MV050506                              10190 Telesis Court, San Diego, CA 92121                                  Page 3 of 3
                               Phone: (858) 457-1919 US Toll-Free: 888-428-0558 Fax: (858) 623-9494
                                                  Website: http:/www.genlantis.com
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Version MV050506                          10190 Telesis Court, San Diego, CA 92121                Page 4 of 4
                           Phone: (858) 457-1919 US Toll-Free: 888-428-0558 Fax: (858) 623-9494
                                              Website: http:/www.genlantis.com

				
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