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LipoD293Protocol.ppt

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					                                    0C
                                    Cat # SL100668 Store at 4
                                                                LipoD293 DNA In Vitro
                                                                Transfection Reagent (Ver. II)
                                                                                                                                     15875 Gaither Drive
                                                                                                                                     Gaithersburg, MD 20877
                                                                                                                                     FAX. 301-560-4919
                                                                                                                                     TEL. 301-330-5966
                                                                    100 l                                                           Toll Free. 1-(866)-918-6812
                                                                    500 l                                                           Email: info@signagenlabs.com
                                                                   1000 l                                                           Web: www.signagenlabs.com

                               This product is for laboratory research ONLY and not for diagnostic use
                                                                                                                  Preparation of LipoD293™-DNA Complex and
                               Introduction:
                                                                                                                  Transfection Procedures
                               LipoD293™ (Ver. II) is an enhanced liposome-based DNA
                                                                                                                  We recommend the LipoD293™ (L):DNA (g)
                               transfection reagent which is specifically formulated and
                                                                                                                  ratio of 3:1 as a starting point which usually
                               optimized for HEK293 cells and other mammalian cells with
                                                                                                                  gives satisfactory transfection efficiency with
                               superior efficiency and invisible cytotoxicity. LipoD293™
                                                                                                                  invisible cytotoxicity. To ensure the optimal size
                               Reagent (Ver. II), 1.0 ml, is sufficient for 300 to 600
                                                                                                                  of complex particles, we recommend using
                               transfections in 24 well plates or 50 to 100 transfections in
                                                                                                                  serum-free DMEM with High Glucose to dilute
                               6 well plates.
                                                                                                                  DNA and LipoD293™ Reagent.
                               Important Guidelines for Transfection:                                             The following protocol is given for transfection in 24-
                               - While the following protocol is for 293 cells, follow the                        well plates, refer to Table 2 for transfection in other
                                 same procedures for other types of mammalian cells. To                           culture formats. The optimal transfection conditions
                                 request protocols for lentivirus production and sf9 insect                       for a majority of adherent cell lines are given in
                                 cell transfection, please email us at info@signagenlabs.com                      the standard protocol described below.
                               - For high efficiency, transfect cells at high density. ~90%                       - For each well, add 0.5 ml of complete medium with
                                 confluency is highly recommended                                                   serum and antibiotics freshly 30~60 minutes before
                               - To lower cytotoxicity, transfect cells in presence of serum                        transfection.
                                 (10%) and antibiotics                                                            - For each well, dilute 1 µg of DNA into 50 µl of
                               Procedures for Transfecting Mammalian Cells                                          serum-free DMEM with High Glucose. Vortex gently
                               1. For Adherent Cells                                                                and spin down briefly.
                               Cell Seeding (see Table 1):                                                        - For each well, dilute 3 µl of LipoD293™ reagent
                               Cells should be plated 18 to 24 hours prior to transfection so                       into 50 µl of serum-free DMEM with High Glucose.
                               that the monolayer cell density reaches to the optimal                               Vortex gently and spin down briefly.
                               ~90% confluency at the time of transfection. Complete                              - Add the diluted LipoD293™ Reagent immediately
                               culture medium with serum and antibiotics is freshly added                           to the diluted DNA solution all at once. (Important: do
                               to each well 30~60 minutes before transfection.                                      not mix the solutions in the reverse order !)
                               Note: For high efficiency, high cell density (~90%                                 - Vortex- mix the solution immediately and spin down
                                      confluency) is essential. High serum levels (>5%)                             briefly to bring drops to the bottom of the tube
                                      usually do not have inhibitory effect on transfection                         followed by incubation of 15~20 minutes at room
                                      efficiency. For some specific cells, maximal transfec-                        temperature to allow DNA-LipoD293™ Reagent
                                      tion efficiencies are observed in presence of serum                           complexes to form.
                                      and antibiotics. We recommend using complete                                  Note: Never keep the DNA/LipoD293™ complex longer
                                      serum/antibiotics-containing medium initially.                                       than 20 minutes
                                                                                                                  - Add the 100 µl LipoD293™/ DNA mixture drop-wise
                               Table 1. A Guideline for Seeding mammalian Cells
                                                                                                                    onto the medium in each well and homogenize the
                               Prior to Transfection in Different Culture Formats
                                                                                                                    mixture by gently swirling the plate.
                                  Culture Dishes                   Surface Area (cm2)   Number of Cells to Seed
                                                                                                                  - Remove LipoD293™/DNA complex-containing medium
                                  T175 Flask                       175                  0.7–1.4 x 107
                                                                                                                    and replace with complete serum/antibiotics containing
 2008 SignaGen Laboratories




                                  T75 Flask                        75                   3.0–6.0 x 106               medium 12~18 hours post transfection. For sensitive
                                  100 mm Dish                      58                   2.2–4.4 x 106               cells, to lower cytotoxicity, remove LipoD293™/
                                  60 mm Dish                       21                   0.9–1.8 x 106               DNA complex and replace with complete medium 5
                                  35 mm Dish                       9.6                  3.5–7.0 x 105               hours after transfection.
                                  6-well Plate                     9.6                  4.0–8.0 x 105             - Check transfection efficiency 24 to 48 hours post
                                  12-well Plate                    3.5                  1.5–3.0 x 105               transfection.
                                  24-well Plate                    1.9                  0.8–1.6 x 105              Storage: LipoD293™ DAN In Vitro Transfection Reagent
                                  48-well Plate                    1.0                  4.0–8.0 x 104              (Ver. II) is stable for up to 12 months at 4 0C. This item
                                  96-well Plate                    0.3                  1.2–2.4 x 104              shipped at ambient temperature
                                    0C
                                    Cat # SL100668 Store at 4
                                                                LipoD293 DNA In Vitro
                                                                Transfection Reagent (Ver. II)
                                                                                                                                  15875 Gaither Drive
                                                                                                                                  Gaithersburg, MD 20877
                                                                                                                                  FAX. 301-560-4919
                                                                                                                                  TEL. 301-330-5966
                                                                    100 l                                                        Toll Free. 1-(866)-918-6812
                                                                    500 l                                                        Email: info@signagenlabs.com
                                                                   1000 l                                                        Web: www.signagenlabs.com

                               This product is for laboratory research ONLY and not for diagnostic use


                               2. For Suspension Cells
                               The following protocol is given for transfection in 24-well plate. The                        - The day before transfection, plating cells in 24-
                               protocol can be scalded up or down according to the surface area of                             well plates at 2x105 cells per well so that they
                               culture vessels.                                                                                      are
                               Cell Seeding:                                                                                   ~90% confluent on the day of transfection. Cells
                               Suspension cells are typically seeded the day of the transfection at a                          are plated in 0.5 ml of their normal growth
                               density of 2 x 105 cells per well (24-well plate). For optimal                                  medium containing serum and without antibiotics.
                               transfection conditions with LipoD293™, seed the number of cells                                Note: High serum levels (>5%) usually do not
                               adapted to the culture vessel format according to Table 3.                                             have inhibitory effect on transfection
                                                                                                                                      efficiency. Sometimes growth medium
                               Table 2. Recommended Amounts for Different Culture Vessel                                              without serum and antibiotics gives the
                                        Formats                                                                                       best efficiency. We recommend replacing
                                Culture Dish                       Transfection   Plasmid       Diluent       LipoD293™               normal serum-containing medium with
                                                                   Volume (ml)    DNA (g)      Volume (mL)   Reagent (L)            serum-free and antibiotics-free medium 30
                                96-well plate                      0.2            0.2           2 x 0.01      0.6                     minutes before transfection.
                                                                                                                             - For each well, dilute 1 µg of DNA into 50 µl of
                                48-well plate                      0.3            0.5           2 x 0.02      1
                                                                                                                               DMEM Serum-free Medium with High Glucose.
                                24-well plate                      0.5            1.0           2 x 0.05      3              - Vortex gently and spin down briefly.
                                6-well plate                       1.0            2             2 x 0.1       6
                                                                                                                             - For each well, dilute 3 µl of LipoD293™ reagent
                                                                                                                               into 50 µl of serum-free DMEM Medium with High
                                35 mm dish                         1.0            2             2 x 0.1       6
                                                                                                                               Glucose. Vortex gently and spin down briefly.
                                60 mm dish                         2.8            5             2 x 0.25      15             - Add the diluted LipoD293™ Reagent to the
                                                                                                                               diluted DNA solution all at once.
                                100 mm dish                        6              7-8           2 x 0.5       21 - 24
                                                                                                                               Note: Do not mix the solutions in the reverse
                                T75 flask                          8              18 - 36       2 x 0.75      54 - 108                order!
                                250 ml flask                       20             50 - 100      2 x 1.25      150 - 300      - Vortex- mix the solution immediately and spin
                                                                                                                               down briefly to bring drops to the bottom of the
                               Table 3. Recommended Number of Suspension Cells to Seed                                         tube followed by incubation of 15 minutes at
                                                                                                                               room temperature to allow DNA-LipoD293
                                Culture Dish                                            Number of Cells
                                                                                                                               Reagent complexes to form.
                                96-well plate                                           2 x 104 – 5 x 104                    - Add the DNA-LipoD293 Reagent complexes (100
                                48-well plate                                           5 x 104 – 1 x 105                      µl) directly to each well and mix gently by
                                                                                                                               rocking the plate back and forth.
                                24-well plate                                           1 x 105 – 2 x 105
                                                                                                                             - Incubate the cells at 37°C in a CO2 incubator
                                6-well plate                                            2 x 105 - 5 x 105                      for 24 ~ 48 hours until they are ready to assay
                                                                                                                               for transgene expression. Alternatively, growth
                                35 mm dish                                              5 x 105 – 2 x 106
                                                                                                                               medium may be replaced after 5 hours without
 2008 SignaGen Laboratories




                                60 mm dish                                              2 x 106 – 5 x 106                      loss in transfection activity.
                                100 mm dish                                             5 x 106 – 1 x 107                      Note: For transfection performed in serum-free
                                                                                                                                      medium, be sure to replace with normal
                               LipoD293™/DNA Complex Preparation and Transfection
                                                                                                                                      growth medium (with serum and
                               procedures: For different cell types, the optimal ratio of
                                                                                                                                      antibiotics) after 5 hours incubation!
                                                                                                                               Scale-up:
                               LipoD293™ (L):DNA (g) varies from 1:1 to 3:1. We
                                                                                                                                In general, for larger size plates, scale up all
                               recommend the LipoD293™ (L):DNA (g) ratio of 3:1 as a
                                                                                                                                amounts, reagents, and volumes in proportion to
                               starting point which usually gives satisfactory transfection
                                                                                                                                the surface area. Some final optimization can
                               efficiency with invisible cytotoxicity. To ensure the optimal size
                                                                                                                                then be made as to what is optimal for the
                               of complex particles, we recommend using serum-free DMEM
                                                                                                                                larger dish size.
                               with High Glucose to dilute DNA and LipoD293™ reagent.

				
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