FuGENE ®
HD Transfection Reagent
1.What this Product Does
Number of Transfection Experiments
In a typical experiment using HeLa or COS-1 cells, 1 ml of FuGENE ®HD Transfection Reagent can be ud to perform up to three hundred transfections in 35-mm tissue-culture dishes, using 3 l of reagent combined with 1 – 2 g DNA per well. This is equivalent to over 6,000wells in a 96-well plate or 1,000 wells in a 24-well plate.
L Optimal expression depends upon experimental conditions includ-ing cell type, passage history, confluence, eding protocol, com-plex incubation time, rum batch, etc. The above amounts of reagents work well with HeLa or COS-1 cells. In other test systems,two- to three-fold higher amounts of reagent yield optimal levels of expression.Formulation
FuGENE ® HD Transfection Reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterile-filtered, and pack-aged in glass vials. It does not contain any ingredients of human
or animal origin.
Storage and Stability
•FuGENE ® HD Transfection Reagent is shipped at +15 to +25°C.•Store FuGENE ® HD Transfection Reagent at +2 to +8°C, with the lid very tightly clod. The reagent is stable through the expiration date printed on the label when stored under the conditions.
L FuGENE ® HD Transfection Reagent remains fully functional even
after repeatedly opening the vial (at least five times over a two-month period) as long as the vial is tightly recapped and stored at +2 to +8°C between us.
L Do not store FuGENE ® HD Transfection Reagent below 0°C.
Components may precipitate and alter results. If you accidently place the reagent at Ϫ20°C, briefly warm it to 37°C to dissolve any precipitate. It should function normally; however, do not return it to the freezer.Special Handling
N Always bring to room temperature and mix FuGENE ® HD Transfec-
tion Reagent prior to u (vortex for one cond or u inversion).N Do not aliquot FuGENE ® HD Transfection Reagent from the original
glass vials. Chemical residues in plastic vials can significantly decrea the biological activity of the reagent. Minimize the contact of undiluted FuGENE ® HD Transfection Reagent with plastic sur-faces.
N Always dilute the reagent by pipetting directly
into rum-free medium. Do not allow the FuGENE ® HD Transfection Reagent to contact the plastic walls of the tube containing the rum-free medium during the dilution step.N Do not u siliconized pipette tips or tubes.
Additional Equipment and Reagents Required
Additional reagents and equipment required to perform transfection assays using FuGENE ® HD Transfection Reagent, but not provided,include:
General Laboratory Equipment
•standard cell culture equipment (e.g., biohazard hoods, incubators,microscope)
•standard pipetters and micropipetters •vortex mixer
For Plasmid Preparation
•purified plasmid stock (0.1 g/l – 2.0 g/l) in sterile TE (10 mM Tris, 1 mM EDTA, pH 8.0) buffer or sterile water
•Genopure Plasmid Midi Kit*, Genopure Plasmid Maxi Kit*, or High Pure Plasmid Isolation Kit* can be ud to prepare plasmid.For Verification of Vector Function •assay appropriate for transfected gene
•G-418* or Hygromycin B* (optional; for stable transfection experi-ments)
For Transfection-Complex Formation
•Opti-MEM I Reduced Serum Medium, water, or rum-free medium •24-well plate to rve as test tube rack for FuGENE ® HD Transfec-tion Reagent vial
•sterile polystyrene tubes or round-bottom 96-well plates
Cells Growing in Log Pha
•lect subconfluent cultures in log pha for preparation of the cell cultures for transfection
•method to quantify cell number to reproducibly plate the same number of cells
Application
FuGENE ® HD Transfection Reagent is a multi-component reagent that forms a complex with DNA, then transports the complex into animal or inct cells. Benefits of FuGENE ® HD Transfection Reagent include:•High transfection efficiency in many common cell types, including HeLa, NIH/3T3, COS-1, COS-7, CHO-K1, Hep G2, HEK-293, MCF7,and some inct cell lines. In addition, you will achieve excellent transfection efficiency in some cell lines (e.g., RAW) that are not transfected well by other reagents. Detailed transfection protocols and sample results are available at .
•Demonstrates minimal cytotoxicity or changes in morphology when adequate numbers of cells are transfected, and eliminates the requirement to change media after the addition of transfection complex.
•Suitable for transient and stable transfection.
•Functions exceptionally well in the prence or abnce of rum;eliminates the need to change media.
To ensure the quality of cells to be transfected, Roche recommends using freshly-obtained, low-passage cell sines form ATCC ®. For more information plea visit and bookmark www.atcc.
For the transient and stable transfection of animal and inct cells
Cat. No. 04 709 691 0010.4 ml (up to 120 transfections)Version November 2007
Cat. No. 04 709 705 001 1 ml (up to 300 transfections)
Store at +2 to +8°C
Cat. No. 04 709 713 001 1)Mega-pack 5 × 1 ml (up to 1,500 transfections)Cat. No. 05 061 369 00110 ml (up to 3,000 transfections)Cat. No. 04 883 560 001
Trial-pack
1)
The five vials are packaged together in one box with one pack inrt
For life science rearch only. Not for u in diagnostic procedures. FOR IN VITRO USE ONLY.
配置清单2.How to U this Product
2.1Before you Begin
Required Amount of FuGENE® HD Transfection Reagent
For initial optimization experiments, transfect a monolayer of cells that is 80 – 90% confluent in a six-well culture dish, using 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2 ratios of FuGENE® HD Transfection Reagent (
l) to DNA (g), respectively. For most cell types, the FuGENE® HD Transfection Reagent:DNA ratios provide excellent transfection levels.
L Subquent optimization may further increa efficiency in your particular application. In addition to varying the volume of FuGENE® HD Transfection Reagent, other parameters may be evaluated (e ction 2.6, Parameters for Optimization, and c-tion 3, Troubleshooting).
Plasmid DNA
•It is critical to accurately determine the plasmid DNA concentration using 260-nm absorption (estimates of DNA content bad on the intensity of gel bands are not sufficiently accurate). Determine the DNA purity using a 260 nm/280 nm ratio; the optimal ratio is 1.8.•Prepare the plasmid DNA solution in sterile TE (Tris/EDTA) buffer or sterile water at a concentration between 0.1 g/l and 2.0 g/l. Cell Culture Conditions
Minimize both intra- and inter-experimental variance in transfection efficiency by using cells that are regularly passaged, proliferating well (best when in a log-growth pha), and plated at a consistent den-sity. FuGENE® HD Transfection Reagent is different from FuGENE® 6 Transfection Reagent regarding the optimal density of cells required for maximal expression with minimal negative effect; F
uGENE®6 Transfection Reagent is formulated to work at low cell densities, whereas FuGENE® HD Transfection Reagent is formulated to work at higher cell densities. Cells must be healthy and free of mycoplasma and other contaminants.
L If you have ud FuGENE® 6 Transfection Reagent in the past, we suggest that you increa the plating density for initial tests using FuGENE® HD Transfection Reagent. For most cell lines, u the reagent at cell-plating densities at least twice that ud with FuGENE® 6 Transfection Reagent to yield maximum protein expression. For most cell lines, cultures should be 80 – 90% con-fluent at the time of transfection. For contact-inhibited cell lines such as NIH/3T3, optimal results are obtained when cells are plated at lower densities.
Other Media Additives
In some cell types, antimicrobial agents (e.g., antibiotics and fungicides) that are commonly included in cell-culture media may adverly affect the transfection efficiency of FuGENE® HD Transfection Reagent. If possible, exclude additives for initial experi-ments. Once high-efficiency conditions have been established, the components can be added back while monitoring your transfection results. Cell growth and/or transfection efficiency may be affected by variations in ra quality or media formulations.
Verification of Vector Function
Optimize transfection conditions with a known positive-control reporter gene construct prior to transfecting cells with a new vector construct:
•Determine transfection efficiency with a reporter gene assay (CAT*,-Gal*, Lucifera*, SEAP*, or hGH*).
•Sequence across the flanking vector inrt regions to verify the integrity of your new construct.2.2Preparation of Cells for Transfection
2.3Overview of Initial Transfection Experiment
Adherent and Suspension Cells in a Six-well Plate or 35-mm Culture Dish
For initial optimization, test FuGENE® HD Transfection Reagent:DNA ratios of 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2 (l for FuGENE® HD Transfection Reagent, and g for DNA, respectively). The preparation of the com-plex for a single well of a six-well plate, or a 35-mm culture dish, is described in ction 2.4. The ratios will function very well for com-monly ud adherent cells and suspension cells. For your particular cell line and culture conditions you may find that ratios of 9:2, 10:2, 11:2, or 12:2 resul
t in even greater expression. Try the ratios if you find the highest expression levels in the 8:2 ratio well.
N Prepare the transfection complex in diluent that does not contain rum (e.g., Opti-MEM I Reduced Serum Medium), even if the cells are transfected in the prence of rum. For some cell lines, the complex may be formed in DMEM or sterile water.
L For additional optimization tips, e ction 2.6 and /fugene/hd
Ratio Overview
Preparation of a transfection complex that is sufficient for a 35-mm culture dish, or one well of a six-well plate, at six different ratios: Tab. 1: Preparation of transfection complex for a 35-mm culture dish.
Cell Type Procedure
Adherent
cells
One day before the transfection experiment, trypsinize
the monolayer, adjust cell concentration, and plate the
cells in the chon cell-culture vesl. For most cell
types, plating 3 – 6 × 105 cells in 2 ml of medium in a
35-mm culture dish (or six-well plate) overnight will
achieve the desired density of Ͼ80% confluency at
the time of transfection. For cell lines with special
characteristics, such as contact-inhibited NIH/3T3
cells, a lower plating density should be ud. If using
culture plates of a different size, adjust the total num-
ber of cells, starting volume of FuGENE® HD Transfec-
tion Reagent, and the starting mass of DNA in
proportion to the relative surface area (Table 2). Suspension
cells
U freshly passaged cells at a concentration of 5×
105/ml to 1 × 106/ml in 2 ml of medium in a 35-mm
culture dish (or six-well plate). If using culture plates
of a different size, adjust the total number of cells,
starting volume of FuGENE® HD Transfection
Reagent, and the starting mass of DNA in proportion
to the relative volume (Table 2).
Tube
label
(ratio)
Diluent
(l)
FuGENE® HD
Transfection
Reagent (l)
DNA
(g)
Comments
3:210032Add the entire volume to
a 35-mm culture dish or
each well of a six-well
plate, or 2 – 15 l to each
well of a 96-well plate.
Suggested volumes for
different culture vesls
are included in Table 2. 4:210042
5:210052
6:210062
7:210072
8:210082
2.4Transfection Procedure
Notes:
L As with any experiment, include appropriate controls. Prepare
wells with cells that remain untransfected, cells with transfection reagent alone, and cells with DNA alone.
L For stable transfection experiments, the complex-containing
烤箱做披萨medium should be left unchanged until the cells need to be pas-saged. At that time, include the appropriate lection antibiotics (G 418* or Hygromycin B*).
L To prepare transfection complexes for different-sized containers or
parallel experiments, proportionally change the quantity of all components according to the total surface area of the cell culture vesl being ud (Table 2).
L For ea-of-u when transfecting small volumes, as in 96-well
plates containing 0.1 ml culture medium per well, prepare 100 l of transfection complex and add 2 – 15 l to each well depending upon the cell type.
L The optimal ratio of transfection reagent:DNA and the optimal
total amount of complex may vary with cell line, cell density, day of assay, and gene expresd.
L After performing the optimization experiment where veral ratios
were tested, lect a ratio in the middle of the plateau for future experiments.2.5
Cotransfection Experiments
Suggestions
For cotransfection experiments with FuGENE ® HD Transfection Reagent, maintain the same total reagent:total DNA ratio as that ud for a single plasmid in your system. Thus, the total amount of the plas-mid DNA should be equal to the amount of plasmid ud in a single plasmid transfection.
ᕡAllow FuGENE ® HD Transfection Reagent, DNA, and
diluent to adjust to +15 to +25°C. Vortex for one cond or invert the FuGENE ® HD Transfection Reagent vial to mix.ᕢDilute DNA with appropriate diluent, for example, Opti-MEM I
Reduced Serum Medium, rum-free medium (without anti-biotics or fungicides), or sterile water to a concentration of 2g plasmid DNA/100 l Opti-MEM (0.02g/l).
L For inct cells, u sterile water as diluent. For other cell
lines, try sterile water or rum-free medium as an alterna-tive diluent.ᕣPlace 100 l diluent, containing 2 g DNA into each of six ster-ile tubes labeled 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2.
Recommendation : U sterile polystyrene tubes or round-bottom, 96-well plates to form the transfection complex.L Due to manufacturer variability with relea agents for
96well plates, we suggest using tissue culture treated 96well plates to reduce variablity.ᕤForm the transfection complex by adding FuGENE ® HD
Transfection Reagent to tubes containing diluted DNA :Pipet the FuGENE ® HD Transfection Reagent (3, 4, 5, 6, 7, or 8l) directly into the medium containing the diluted DNA with-out allowing contact with the walls of the plastic tubes.
N To avoid adverly affecting transfection efficiency, do not摘棉花
allow undiluted FuGENE ® HD Transfection Reagent to come into contact with plastic surfaces (such as the walls of the tube that contains the rum-free medium) other than pipette tips. Do not u siliconized pipette tips or tubes.ᕥMix and incubate the transfection complex :
Vigorously tap the tube or vortex for one to two conds to mix the contents. If using a 96-well plate, place the plate on a rotat-ing shaker for 5 – 10 conds. Incubate the transfection reagent:DNA complex for 15 minutes at room temperature.For some ratios and cell types, incubation is not necessary for optimal complex formation, while a longer incubation time is better for other cell types. Determine this for your particular cell line and the ratio you u.ᕦAdd the transfection complex to cell s:
Remove culture vesl from the incubator. Removal of growth medium is not necessary. Add the transfection complex to the cells in a drop-wi manner or add below the surface of the medium. Swirl the wells or flasks to ensure distribution over the entire plate surface. U of a rotating platform shaker for 30 conds at low speed provides adequate mixing for 96-well plates.
Once the FuGENE ® HD Transfection Reagent:DNA complex has been added to the cells, there is n
应急金o need to remove and replace with fresh medium (as is necessary with some other transfection reagents).
L In our experience, the exposure of most common laboratory
cell types (COS-1, CHO-K1, HEK-293, HeLa, Hep G2, MCF-7) to the transfection complex until performance of the gene expression assay (24–48 hours later) does not affect the results. If you desire to transfect cells that are in rum-free medium during the transfection process, then replace the medium with rum-containing medium 3 – 8 hours after transfection, unless the cells normally grow in rum-free medium. ᕧIncubate cells and assay the results :
Following transfection, incubate the cells for 18 – 72 hours prior to measuring protein expression. The length of incubation depends upon the transfected vector construct, the cell type being transfected, the cell medium, cell density, and the type of protein being expresd. After this incubation period, measure protein expression using an assay that is appropriate for your system.
L If you obrve low transfection levels or more than
10 – 30% cell death, refer to ction 3, Troubleshooting /fugene/hd
2.6Parameters for Optimization
2.7Transfection of Adherent Cells Adapted for Suspension Growth加湿器英文
•In some cas, adherent cells may be adapted for suspension growth, thus enabling the production of transiently transfected cells on a very large scale.
•HEK-293 cells grown in suspension in rum-free medium that did not contain heparin or dextran sulfate produced significant amounts of protein following transfection.
2.8Guidelines for Preparing FuGENE® HD Transfection Reagent:DNA Complex for Various Culture Vesl Sizes
The starting volume and mass to add to the different culture vesls is bad upon preparing a 100-l transfection complex as described in c-tions 2.3 and 2.4. For best results, prepare a 100-l complex at different ratios and add varying amounts of each ratio when optimizing. The amounts below are bad on the 100-l complex as prepared in ctions 2.3 and 2.4.
Suggested eding density for adherent cells = 30,000 – 70,000 cells per cm2
Suggested eding density for suspension cells = 250,000 – 500,000 cells per ml
Tab. 2: Refer to the table below when tting up your transfection reac-tions. T he are suggested eding densities and are media, passage level, laboratory, and cell-line dependent. It is critical that log pha cultures are lected for subculture for the transfection experiments, and that cultures are eded at the proper density for the transfection experiment. Obrve cultures and plate them so that the monolayer is 80–90% confluent at the time of trans-fection. This must be determined empirically. For some cell lines, 60–80% con-fluency is sufficient. However, a contact-inhibited cell line, such as NIH/3T3, should be plated at lower confluence due to its growth characteristics.1) Scale up total volume for larger vesls.
3.Troubleshooting
Parameter to be
好看的日剧恋爱剧optimized
Procedure
FuGENE® HD Transfection Reagent:DNA ratio Form the transfection complex at veral ratios: 3:2, 4:2, 5:2, 6:2, 7:2, 8:2, 10:2, and 12:2 (l FuGENE® HD Transfec-tion Reagent: g DNA).
In some systems, altering the ratio of FuGENE® HD Transfection Reagent to DNA can increa the level of protein expression.
L It has been reported that for some plasmid preparations, a ratio of 2:2 yielded optimal results. This is unusual and may reflect some property of the plasmid preparation rather than a characteristic of the FuGENE® HD Transfec-tion Reagent.
Amount of transfection
complex added
Try adding 200%, 150%, 75%, 50%, and 25% of the amount of 100-l transfection complex suggested in Table 2.
Number of cells plated Plating more cells will overcome negative growth effects of excess transfection complex. For cells with special growth characteristics, such as NIH/3T3 cells, do not u this as the first parameter for optimization.
Incubation time for the transfection complex to form Vary the length of incubation time for transfection-complex formation: add the complex to the cells immediately after the components are c
ombined and mixed, and then at veral intervals up to 40 minutes (i.e., 0, 15, 25, and 40 min-utes). We have obrved that in some cell lines, the transfection-complex incubation time tends to have no effect on results when using higher ratios; however, results using lower-ratio-complexes varied depending on the incubation time for complex formation.
Special tips for nsitive cell lines •Reduce the time of exposure to the transfection complex (2–3 hours maximum), then replace the medium.•U the lower ratios, and allow the complex to form for a longer period of time (determine empirically for your cell line), then add lower amounts of the complex (50% or less of what was originally tested).
Culture vesl Surface
area
(cm2)
Total
volume
of
medium
Suggested eding density
Suggested amount of
the 100-l trans-
fection complex to
add to each well (l)
Final amount of FuGENE® HD
Transfection Reagent (l) in
each well following addition of
suggested amount of 100-l
transfection complex
Cells/well
Adherent cells
Cells/well
Small or suspension
cells
Using the
3:2 ratio
Using the
8:2 ratio total
volume
volume for
larger
low high low high
96-well plate
(1 well)
0.30.110,00020,00025,00050,00050.150.4
24-well plate
(1 well)
1.90.550,000125,000250,000500,000250.75
2.0
12-well plate
(1 well)
3.8 1.0100,000250,000375,000750,00050 1.5
4.0 35-mm dish82200,000500,000500,0001,000,000100 3.08.0
6-well plate
(1 well)
9.42200,000600,000500,0001,000,000100 3.08.0 60-mm dish215500,0001,400,0001,250,0002,500,000250 1)7.520.0 10-cm dish55101,500,0003,500,0002,500,0005,000,000500 1)15.040.0 T-25 flask256700,0001,700,0001,500,0003,000,000300 1)9.024.0 T-75 flask75202,000,0005,000,0005,000,00010,000,000900 1)27.072.0
Obrvation Possible Cau Recommendation
Low trans-fection efficiency Poor quality or
insufficient quantity
of nucleic acids
Verify the amount, purity, and quence of nucleic acid.
Perform a control transfection experiment with a commercially available transfection-grade plasmid
preparation.
Chemical contaminants may be in the plasmid preparation. Avoid phosphate buffers until you have
tested them in your system.
L Endotoxins are reported to be cytotoxic to some very nsitive cell lines.
Insufficient number
of cells
U adherent cells that are at least 80% confluent. Low cell density results in fewer cells available to
take up transfection complex, and excess complex may be cytotoxic; in addition, fewer cells yield
less protein.
Too many cells or
cells post log pha
When confluent cultures are subcultured, or cells are plated at too high a density, the cells fail to divide
in the culture being transfected. This results in suboptimal expression.
Suboptimal FuGENE®
HD Transfection
Reagent:DNA ratio,
complex incubation
time, total amount of
transfection complex
added, or cell density
Optimize the FuGENE® HD Transfection Reagent:DNA ratio, complex incubation time, amount of
complex added to cells, and cell density, according to the following procedure:
Day before transfection:
Prepare two 96-well plates of cells at high and low eding densities (e Table 2 for suggestions).
Day of transfection:
•Form 200 l of transfection complex at ratios of 2:2, 3:2, 4:2, 5:2, 6:2. 7:2, and 8:2 (l transfection
reagent:g DNA) following the protocol in this pack inrt (ctions 2.3, 2.4) and doubling the
amounts of all components.
联盛超市
•As soon as the complexes are combined and mixed, add 10, 5, or 2.5 l of each complex to one of
3columns of cells in each 96-well plate (i.e., columns 2, 3, and 4). Leave all outer wells empty as
controls.
•Continue to incubate the complexes at room temperature. After an additional 10 – 15 minutes, add 10,
5, or 2.5 l of each complex to the next 3 columns (5, 6, and 7) of cells in each 96-well plate.
•Continue to incubate the complexes at room temperature. After an additional 10 – 15 minutes, add 10,
5, or 2.5 l of each complex to the next 3 columns (8, 9, and 10) of cells in each 96-well plate.
•Assay the plates 1–2 days later. Select the ratio, amount of complex, and time of transfection-complex
incubation that resulted in optimal expression.
•If optimal transfection occurs at the higher ratios, repeat this process using ratios of 6:2, 7:2, 8:2, 10:2,
12:2, and 14:2. Add 5, 10, and 15 l of complex. We have never successfully transfected cells using the
ratio of 2:2, but it has been reported that some plasmid preparations transfect at this ratio.
See ction 2.6, Optimization of FuGENE® HD Transfection Reagent:DNA ratio, for more information and
/fugene/hd
FuGENE® HD Trans-
fection Reagent was
aliquoted
Check that FuGENE® HD Transfection Reagent is stored in the original container. If the reagent was
aliquoted into plastic containers, there is a high chance of inactivation. Make sure the reagent isgeexxx
immediately mixed with the dilute DNA either by vortexing or pipetting up to 10 – 15 times.
FuGENE® HD Trans-
fection Reagent came
into contact with
plastic or was
inadequately mixed
Repeat transfection, carefully pipetting FuGENE® HD Transfection Reagent directly into the rum-free
medium, being careful not to touch the sides of the container while adding the FuGENE® HD Transfec-
tion Reagent to the diluted DNA. If the FuGENE® HD Transfection Reagent is added too gently, it may
layer on top of the medium, thus making contact with the plastic.
Transfection complex
was formed in rum-
containing medium
Check original bottle of medium ud for complex formation. Repeat experiment using new bottle of
Opti-MEM that does not contain any additives (e.g., rum, antibiotics, growth enhancers, heparin,
dextran sulfate, etc.). Try forming the complex in sterile water or plain DMEM.
Media and media
components
Different media and media components may influence the level of transfection efficiency and
subquent growth of the transfected cells, as well as expression of the recombinant protein. Some lots
of ra have been reported to interfere with optimal transfection.
Quality and/or lot-to-lot differences that affect transfection experiments have been noted in both ra
and media. Check that the medium and/or rum is from the same lot that worked previously. Try new
lots or a different vendor.
Culture may be
contaminated with
mycoplasma
Cultures contaminated with mycoplasma have been shown to have decread transfection efficacy.
Determine if culture is contaminated with mycoplasma; u the Mycoplasma Detection Kit* or
Mycoplasma PCR ELISA* to asss contamination.
