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fugene 6转染试剂

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For life science research only. Not for use in diagnostic procedures. FuGENE 6

Transfection Reagent

Multi-component formulation for the transfection of eukaryotic cells Cat. No. 1 815 0910.4 ml(80–120 transfections) Cat. No. 1 814 443 1 ml(200–300 transfections) Cat. No. 1 815 075Multi-pack 5 x 1 ml(1000–1500 transfections) Cat. No. 1 988 484Custom pack Inquire(10 ml or 50 ml glass vials) Store FuGENE 6 Transfection Reagent at –15 to –25oC.

Instruction Manual

Version 5, September 2000

1. Preface

1.1 Table of contents

1.Preface (2)

1.1Table of contents (2)

2.Product characteristics (3)

3.Introduction (3)

3.1Product overview (3)

3.2Background information (4)

4.Procedures and required materials (5)

4.1Before you begin (5)

4.2Factors for successful transfection (5)

4.3Standard protocol for transient and stable transfection (8)

4.3.1Preparation of cells for transfection (8)

4.3.2Preparation of FuGENE 6 Reagent:DNA complex (9)

4.3.3Scale-up for large transfection experiments (9)

4.3.4Transfection of cells (10)

4.3.5Cotransfection experiments (10)

4.3.6Optimization of transfection efficiency and protein expression levels (10)

4.3.7Measurement of protein expression (11)

4.4 Large-scale transfection using FuGENE 6 Reagent for

transient protein expression (11)

4.4.1 Optimization in high through-put screening (HTS) applications and

protein production (12)

5.Appendix (13)

5.1Troubleshooting table (13)

5.2How to contact Roche Molecular Biochemicals (16)

5.3 References

(17)

5.4Related products...............................................................................................................................18P R O C E D U R E S

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2. Product characteristics

Formulation FuGENE 6 Transfection Reagent is a proprietary blend of lipids and

other components supplied in 80% ethanol, sterile-filtered, and

packaged in polypropylene tubes. Warm FuGENE 6 Transfection

Reagent to ambient temperature (approximately 10 to 15 minutes at

room temperature) prior to use. Always mix FuGENE 6 Reagent prior to

use (vortex or inversion). After long periods of storage, FuGENE 6

Transfection Reagent may be slightly turbid due to the precipitation of

some components. In most systems, the turbidity has no effect on the

biological activity of the product.

NOTE: The new Custom-pack of FuGENE 6 Transfection Reagent

is supplied in glass vials.

Storage and stability FuGENE 6 Transfection Reagent is stabilized for shipping at room temperature and for extended storage at –20°C through the expiration date printed on the label (two years from date of manufacture). FuGENE 6 Reagent is shipped to you at room temperature.

Special handling Do not aliquot FuGENE 6 Reagent from the original polypropylene tubes. Chemical residues in new plastic vials can significantly decrease the biological activity of the reagent.

NOTE: Evaluations have shown that FuGENE 6 Transfection Reagent remains fully functional even when vials are repeatedly opened (at least 6 times over a 3 month period), as long as the vials are recapped tightly and stored at –20°C between use. A 20% loss of ethanol due to evaporation does not substantially affect biological activity in COS-1 cells.

3. Introduction

3.1 Product overview

Application FuGENE 6 Transfection Reagent efficiently transfects a wide variety of mammalian cells and other cell types with high efficiency. Visit the

FuGENE 6 Reagent web page for a current list of >250 successfully

transfected cells lines: http://biochem. https://www.wendangku.net/doc/0818264495.html,/techserv/fugene.htm Number of tests One milliliter of FuGENE 6 Reagent transfects approximately 200–300,

35 mm tissue culture dishes (total media volume of 2 ml each) with

one of the following cell lines (HeLa, NIH 3T3, COS-1, COS-7,

CHO-K1).

NOTE: The levels of expression of the gene of interest will vary

with the cell line.

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3.1 Product overview, continued

Quality control Functional analysis Three microliters of FuGENE 6 Transfection Reagent is combined with 1–2 μg of a reporter gene vector DNA, and used to transfect COS-1 cells in a monolayer (50–80% confluent) in the presence of 10% fetal bovine serum (FBS). Following transfection, the percentage of cells transfected is analyzed, and typically, 50%–70% of COS-1 cells express reporter gene protein.

Cytotoxicity analysis COS-1 cells continuously exposed for 26 hours to FuGENE 6 Reagent,

with or without DNA, in the presence of serum, and without a change of medium, are >90% viable by flow cytometric analysis based on

propidium iodide-staining.

3.2 Background information

General transfection methods Transfection is the general process of bringing foreign DNA into cells and monitoring protein expression. DNA transfection is essential for the study of gene function and gene regulation.

?Common transfection techniques include calcium phosphate coprecipitation (1), electroporation (2,3), and the use of viral vectors

(4). These methods have produced variable results in a variety of

cell types (5–9).

?Cationic liposome-mediated transfection methods (lipofection, cytofection) were an important addition to the previous methods

(10). Additional classes of compounds known to mediate

transfection include lipopolyamines (11) and dendrimers (12).

FuGENE 6 Transfection Reagent FuGENE 6 Transfection Reagent is a multi-component lipid-based transfection reagent that complexes with and transports DNA into the cell during transfection. Benefits of FuGENE 6 Reagent include:

?Provides very high transfection efficiency in many common cell types.

?Demonstrates virtually no cytotoxicity even in many primary cell types.

?Functions exceptionally well in the presence or absence of serum.

?Requires minimal optimization.

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4. Procedures and required materials

4.1 Before you begin FuGENE 6 Reagent handling

NOTE: FuGENE 6 Transfection Reagent is unique as compared to other liposomal transfection reagents, and requires special handling. Review the following steps before proceeding further.

4.2 Factors for successful transfection Overview

The following factors affect transfection reactions:?FuGENE 6 Reagent working concentration ?Concentration and purity of nucleic acids ?Cell culture conditions

?Transfection in serum-containing or serum-free media ?Other media additives

?Verification of vector function ?High protein expression levels

continued on next page

Special handling steps

Always dilute FuGENE 6 Reagent by pipetting directly into

serum-free medium. Do not allow the undiluted reagent to come into contact with any plastic surface. Refer to section 4.3.2, Preparation of FuGENE 6 Reagent:DNA complex.

2Always use more FuGENE 6 Reagent (μl volume) than DNA (μg mass). For details, see section 4.2.

3Always store in the original polypropylene tubes. Do not aliquot. Chemical residues in new plastic vials can significantly decrease the biological activity of the reagent.

4FuGENE 6 Reagent is very robust, and unlike other reagents, transfects most cell types equally well in the presence or absence of serum, reducing the number of handling steps.

For most cell types: All additional handling steps after the

addition of the FuGENE 6 Reagent:DNA complex to the cells can be eliminated.

Since FuGENE 6 Reagent is virtually nontoxic and transfects in the presence of serum, the transfection complex can be added to cells in serum-containing growth medium without washing (section 4.2). Additionally, the complex can be left on the cells until the time of the protein expression assay.

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4.2 Factors for successful transfection , continued

FuGENE 6 Reagent working concentration For an initial experiment in a 35 mm culture dish, containing a mono-layer of cells that is 50–80% confluent, transfect cells using FuGENE 6 Transfection Reagent (μl) to DNA (μg) amounts of 3:2, 3:1, and 6:1,

respectively (refer to section 4.3.2 for important details of FuGENE 6:DNA complex preparation). For most cell types, these FuGENE 6 Reagent:DNA amounts provide excellent levels of transfection.

NOTE: Subsequent optimization may further increase efficiency in your particular application. Hold the amount of DNA constant while increasing the volume of FuGENE 6 Reagent (as illustrated in Figure 2). Transfection efficiency is greatly reduced when the amount of DNA exceeds a ratio of 3:2 (volume FuGENE [μl]): mass DNA [μg], see Figures 1 and 2). Concentration and purity of nucleic acids

It is critical to accurately determine the concentration of your DNA using 260 nm absorbance. For initial experiments, avoid possible cytotoxic effects by using highly purified nucleic acids (e.g., cesium chloride gradient or column purified), free of traces of residual cesium chloride. Once transfection conditions are established, plasmid prepa-ration with higher endotoxin levels can be tested. NOTE: Use a plasmid concentration between 0.02 and 2.0 μg/μl.

Cell culture conditions

Use cells that are healthy, proliferating well, and plated at a consistent density to minimize both intra- and interexperimental variance in transfection efficiency.

NOTE: Use only regularly passaged cells in a log-growth phase.

P R O C E D U R E S

Figure 1. Effect of DNA amount on transfection efficiency using FuGENE 6 Transfection Reagent.COS-1 cells (1.3 x 105) were plated in 35 mm dishes 18 hours prior to transfection. Cells were transfected with one of three fixed volumes of FuGENE 6 Reagent and various quantities of pCMV-SEAP plasmid.Expression of the SEAP reporter gene was deter-mined with the chemiluminescent SEAP Reporter Gene Assay (Cat. No. 1779842).

Figure 2. Effect of the presence or absence of serum on transfection efficiency with FuGENE 6Reagent. COS-1 cells (1.3 x 105) were plated in 35mm dishes. The next day, different volumes of FuGENE 6 Reagent were used to transfect cells with 2 μg pCMV-b Gal in the presence or absence of serum. Cells were lysed using 0.25 ml of lysis buffer,and β-galactosidase expression was determined by analyzing 5μl cell lysate with the chemiluminescent β-Gal Reporter Gene Assay (Cat. No. 1758241).

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4.2 Factors for successful transfection, continued

Transfection in serum-containing or serum-free media FuGENE 6 Reagent produces highly efficient transfection both in the presence and absence of serum (Figure 2). In some cell types, transfection efficiency may improve in the presence of serum, and in other cell types, higher transfection efficiency may be achieved in reduced-serum medium, or under serum-free conditions. The same type of cell culture medium routinely used for culturing cells can also be used during transfection.

If low or reduced serum conditions are used for transfection, return cells to normal serum concentrations by adding additional serum or changing the medium 3–8 hours post transfection.

NOTE: Prepare the FuGENE 6 Reagent:DNA complex in medium that does not contain serum.

Other media additives In some cell types, antimicrobial agents (e.g., antibiotics and fungicides) commonly included in cell culture media, have been observed to adversely affect the transfection efficiency of FuGENE 6 Transfection Reagent. Exclude additives for initial experiments if possible. Once high-efficiency conditions have been established, these components can be added, however, you will need to monitor your transfection results.

Verification of vector function Prior to transfecting cells with a new vector construct, verify and (if necessary) optimize transfection conditions with a known positive control reporter gene construct.

?Use the same vector backbone as your construct, where a reporter gene was previously inserted.

?Determine the transfection efficiency by measuring the level of reporter protein expression with a reporter gene assay (CAT, β-Gal, luciferase, SEAP, or hGH, see section 5.4, Related products).

?Confirm insert size by performing PCR with the Intelli-Search Bacterial Colony Screen Kits (section 5.4, Related products).?Sequence across the flanking vector insert regions to verify the integrity of your new construct.

High protein expression levels High levels of expression of certain proteins (e.g., GFP) may be

cytotoxic for some cell types. Refer to section 5.1, Troubleshooting

guide.

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4.3 Standard protocol for transient and stable transfection Additional required reagents

?Sterile, serum-free culture medium, (optional: add 12.5 mM HEPES buffer to serum-free medium).

?DNA stock solution in 1x sterile TE buffer or sterile water with a concentration determined by 260 nm absorption.

4.3.1 Preparation of cells for transfection Adherent cells

Plate the cells one-day before the transfection experiment. The appropriate plating density will depend on the growth rate and the condition of the cells. Use cells that are 50–80% confluent on the day of the experiment. Plating most cell lines at 1–3 x 105 cells in 2 ml in a 35 mm culture dish (or 6-well plate) will achieve this density after overnight incubation.

NOTE: Adjust the number of cells accordingly if using culture plates of different sizes (see Table 1 below).

Suspension cells

Use freshly passaged cells at a concentration between 5 x 104/ml to 1x 106/ml (2ml in a 35 mm culture dish or 6-well plate). Determine cell number based on your needs and cell type to be transfected. NOTE: It is usually not necessary to wash the cells prior to the addition of the transfection reagent:DNA complex because

performance is independent of the presence or absence of fetal bovine serum in the cell culture medium (Figure 2).

Table 1

Refer to the following table when setting up your transfection reac-tions. This table is based on a transfection reagent:DNA ratio of 3:1. Refer to section 4.3.2 to use other ratios.Type of dish or plate Surface area per well or

plate (cm 2)Total media volume per well or plate (ml)

Starting volume of FuGENE 6 Reagent (μl/well or plate)

Starting mass of DNA (μg/well or plate)60 mm 214 6.0 2.035 mm 82 3.0 1.06-well 9.42 3.0 1.012-well 3.81 1.50.524-well 1.90.50.60.296-well

0.3

0.1

0.15

0.05

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4.3.2 Preparation of FuGENE 6 Reagent:DNA complex

Adherent and suspension cells in a 35 mm culture dish The following ratios have been optimized for adherent cells. These ratios also work with suspension cells.

Use FuGENE 6 Reagent:DNA amounts of 3:2, 3:1, and 6:1 (μl and μg, respectively) in each well of a 6-well plate or 35 mm culture dish (described below).

NOTE: To further optimize conditions, test a broader range

(2–15 μl) of FuGENE 6 Transfection Reagent, while keeping the amount of DNA constant at 1–2 μg. Refer to section 5.1, Troubleshooting.

4.3.3 Scale-up for large transfection experiments

Preparation of master mix complex To prepare transfection complexes using the same DNA for many

parallel experiments, proportionally increase the quantity of all components, including the serum-free medium, FuGENE 6

Transfection Reagent, and the vector DNA.

Step Preparation of complex

1In a small sterile tube, add sufficient serum-free medium as

diluent for FuGENE 6 Transfection Reagent, to a total volume of

100 μl. Add 3 to 6 μl of FuGENE 6 Reagent directly into this

medium. The order of addition is critical. The serum-free

medium must be pipetted into the tube first. Tap gently to mix.

NOTE: To avoid adversely affecting transfection efficiency,

do not allow undiluted FuGENE 6 Reagent to come in

contact with plastic surfaces other than the pipette tip.

2Add 1–2 μg DNA solution (0.02–2.0 μg/μl) to the prediluted

FuGENE 6 Reagent from Step 1. Use a total volume of DNA

solution between 0.5–50μl.

3Gently tap the tube to mix the contents. DO NOT VORTEX.

Incubate for a minimum of 15 minutes at room temperature.

Continued incubation for up to 45 minutes (for some cell lines

up to 2 hours) will not affect the transfection efficiency in most

cell types. Go to section 4.3.4, Transfection of cells.

FuGENE 6

Reagent:DNA ratio

FuGENE 6 Reagent

volume (μl)

DNA volume

(μg)

3:2 3 2

3:131

6:1 61

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4.3.4 Transfection of cells Transfection procedure

The following procedure has been optimized for adherent cells (Step 1). Suspension cells can be transiently transfected using the same procedure after transfer to a new plate or flask. 4.3.5 Cotransfection experiments Suggestions

Cotransfection experiments with FuGENE 6 Reagent have been performed simultaneously using up to seven different plasmids. Increase the amount of transfection reagent in proportion to the

amount of total μg DNA when performing cotransfection experiments. NOTE: Always use excess volume of FuGENE 6 Reagent over the total final mass of DNA (section 4.2).

4.3.6 Optimization of transfection efficiency and protein expression levels Optimization factors

Consider the following factors when optimizing your transfection reaction.

?FuGENE 6 Reagent:DNA ratio ?Cell density and growth phase ?Cell passage history

?Number of hours to measurement of reporter gene activity

Step Transfection procedure

1Dropwise, add the complex mixture from Step 3 (section 4.3.2) to your cells, distributing it around the well. Swirl the wells or flasks to ensure even dispersal.

Return the cells to the incubator until the time of the reporter gene assay.

NOTE: There is no need to remove the reagent:DNA complex from the cells prior to the reporter gene assay. In our experience, exposure of most common laboratory cell types (COS-1, CHO-K1, HEK-293, HeLa) to the reagent: DNA complex until the time of the reporter gene assay (24–48 hours later), has

produced no adverse effects, however, this may need to be determined for your particular cell type. If you observe cytotoxicity with the FuGENE 6:DNA complex, refer to section 5.1, Troubleshooting. For stable transfection experiments, the complex

containing medium can be left unchanged until the cells need to be fed.

3 (optional)

Use serum-free medium during the transfection procedure, and replace the medium with serum-containing medium 3–8 hours after transfection, or add serum directly to wells.

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4.3.7 Measurement of protein expression

Variables Incubate the cells 4–72 hours. The length of incubation depends upon the transfected vector construct, the cell type being transfected, and

the protein being expressed. After this incubation period, measure

protein expression using an assay appropriate for your system.

4.4 Large-scale transfection using FuGENE 6 Reagent for

transient protein expression

Overview?FuGENE 6 Reagent has been successfully used to greatly increase

the speed of drug discovery in pharmaceutical drug screening

programs. This is accomplished by eliminating the need to establish

stable cell lines expressing receptors or ligands of interest.

?Large-scale transient transfection experiments using FuGENE 6

Reagent yield cells expressing molecules of interest at high levels.

The exceptionally low toxicity of the reagent is especially beneficial.

After transfection, cells retain most normal physiological functions,

and can therefore serve as targets for various screening applica-

tions. In addition, several cumbersome steps required with first

generation transfection reagents can be eliminated with the use of

FuGENE 6 Reagent (see Table 2). As a result, you can easily screen

thousands of small molecules within a very short time period using

transiently transfected cells.

?FuGENE 6 Reagent has also been used to express pharmacologi-

cally interesting receptors at high density in cells, followed by the

preparation of membrane fractions for classical drug binding

studies (unpublished data). In more complex studies, cells can be

transfected with molecules of interest, and then the whole cells can

be used in screening assays while monitoring physiological activity

with specific indicators.

Table 2.

Step FuGENE 6

Transfection

Reagent First generation transfection

reagents

Count and plate cells in FBS medium÷÷Incubate overnight÷Remove FBS-containing media÷Replace media without FBS÷Add transfection reagent:DNA

complex

÷÷

Remove transfection reagent:DNA

complex after 4–6 h

Replace with fresh serum-containing

media

÷Total transfection steps27P R O C E D U R E S

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4.4.1Optimization in high through-put screening (HTS) applications and

protein production Optimization of large-scale transfections

Refer to the following table for a list of factors that can shorten the transfection procedure, and help attain maximal levels of protein expression.

Optimization of FuGENE 6 Reagent/DNA ratio ?In some systems, increasing the amount of both FuGENE 6 Reagent and DNA (more than ten fold higher than the recommended amounts), can continue to increase the level of protein expression.

?The very low cytotoxicity of FuGENE 6 Reagent permits both the FuGENE 6 Reagent and DNA levels to be tested at these high levels without adversely affecting cell viability.

Transfection of cells immedi-ately following trypsinization ?Use FuGENE 6 Reagent to transfect some cell lines immediately following trypsinization and just prior

to or after plating. ?This will substantially reduce set-up time by

eliminating the need to wait 24 hours before transfection.

Transfection of adherent cells adapted for suspension growth ?In some cases, adherent cells may be adapted for suspension growth, reducing requirements for expensive sterile plastic tissue culture vessels, and enable the production of transiently transfected cells on a very large scale.

?HEK-293 cells have been transfected while in suspension growth using FuGENE 6 Reagent (unpublished data).

Effect of media and media components, including sera ?Different media and media components may influence the level of transfection efficiency and the subsequent growth of the transfected cells, as well as expression of the recombinant protein.

?Test different media and optimize the level of each medium component for these effects. Although it is not usually necessary to remove the transfection reagent:DNA complex following the transfection step, it is necessary to feed your cells with fresh media for extended growth periods. This is especially important if the transfected cells are allowed to continue to grow for 3–7 days, allowing for maximal protein expression.

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5. Appendix

5.1 Troubleshooting table

Low transfection efficiency

Refer to the following table if you observe a low transfection efficiency.continued on next page

Problem Possible cause Recommendation

Low transfection efficiency Nucleic acids of poor quality or insufficient quantity

Verify the amount or quality of nucleic acid:?Use only high-quality plasmid preparations, see

section 4.2.?Use DNA at a concentration of 0.02–2.0 μg/μl.

?Verify that the transfected plasmid construct contains appropriate promoters and other sequences required for protein expression in the cell line being transfected.?Perform a control transfection experiment with a commercially available transfection-grade plasmid preparation (e.g., the β-gal control vectors supplied with the Mammalian Expression Vectors for Epitope Tagging (Cat. No. 1 814 664).

NOTE: Endotoxins are reported to be cytotoxic to some very sensitive cell lines (e.g., Huh-7) and primary cultures (13). When using FuGENE 6 Reagent for many common cell types, it may be possible to use DNA containing higher endotoxin levels.

Insufficient number of cells were used Use adherent cells at 50–80% confluency. FuGENE 6 Reagent was aliquoted and stored in a new container Check that FuGENE 6 Reagent is stored in the original container.FuGENE 6 Reagent came into contact with plastic

Repeat transfection, carefully pipetting FuGENE 6 Reagent directly into the serum-free https://www.wendangku.net/doc/0818264495.html,plex was formed in serum-containing medium Check original bottle of medium used for complex formation. Repeat experiment using new bottle of medium that does not contain any additives (e.g., serum, antibiotics, growth enhancers, etc.)

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5.1 Troubleshooting table , continued

continued on next page

Problem Possible cause Recommendation Low transfection efficiency A suboptimal FuGENE 6:DNA ratio was used ?Optimize the FuGENE 6 Reagent:DNA ratio according to the following procedure. Note: Always use more

FuGENE 6 Reagent (μl) than DNA (μg). For

example, combine 3 μl FuGENE 6 Reagent with 1–2 μg DNA for a 35 mm culture dish (6-well plate).

?Prepare FuGENE 6:DNA mixtures according to the following table. Do not allow FuGENE 6 Transfection Reagent to come in contact with the plastic tube before dilution with serum-free medium.

1Tap the tubes gently. Mix thoroughly, but do not vortex.

2Incubate at room temperature for 15-45 minutes.3Add each FuGENE 6 Reagent:DNA mixture to a

35mm culture dish or one-well of a 6-well plate. Swirl the plates.

4If you raise the DNA concentration (e.g., in a cotrans-fection experiment), proportionally increase the amount of FuGENE 6 Transfection Reagent.

?If your cell line is not easily transfected by the above FuGENE 6 Reagent:DNA ratios, test a wider range of ratios, including 2–15 μl FuGENE 6

Transfection Reagent per 1–2 μg DNA, per 35 mm culture dish.

?If no transfection is observed, repeat the

experiments with DOTAP or DOSPER Liposomal Transfection Reagent (see section 5.4, Related products).

Label six

tubes 123456Add serum-free media (μl) 979797949494Add FuGENE 6 Reagent (μl)3

3666Add DNA

(μg)

0.51

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5.1 Troubleshooting table , continued

Signs of cytotoxicity Refer to the following table if you observe signs of cytotoxicity.

continued on next page

Problem Possible cause Recommendation

Signs of cytotoxicity

NOTE: FuGENE

6 Transfection Reagent has

proven to be

virtually non-toxic to most cell types. Selection anti-biotic added too soon

Repeat transfection and wait an additional 24 to 48 h before adding the selection antibiotic, to allow for sufficient protein production.

Selection anti-biotic at too high a concentration Repeat transfection using several lower concentrations of selection antibiotic.

Transfected

protein is cyto-toxic or is pro-duced at high

levels

Reduced viability or slow growth rates may be the result of high levels of protein expression, as the cells ’ meta-bolic resources are directed toward production of the heterologous protein. The expressed protein may also be toxic to the cell at the level expressed.

To analyze cytotoxicity, prepare experimental controls as described.

Prepare extra wells containing:a. Cells that are not transfected.

b. Cells transfected with DNA alone (e.g., without FuGENE 6 Transfection Reagent)

c. Cells treated with FuGENE 6 Reagent alone (no DNA added).

?Compare transfected cells with the experimental construct, to the wells containing these experimental controls.

?Consider repeating the experiment with a secreted reporter gene assay such as SEAP, hGH, or a standard β-gal control vector (see low transfection efficiency above). Cells secreting SEAP should show little to no evidence of cytotoxicity.

The culture may be contaminated with mycoplasma

?Use the Mycoplasma Detection Kit or Mycoplasma PCR ELISA (see section 5.4) to determine if the culture is contaminated.

?Treat the cells with BM Cyclin to eliminate the myco-plasma. Alternatively, start the transfections over with a fresh uncontaminated culture.

Cells may not be healthy (e.g., malfunctioning incubator, media problems)

Assess physiological state of cells and the incubation conditions (e.g., CO 2 and temperature levels). Perform the same controls as suggested above (for cytotoxicity), to eliminate influence of transfection reagent or nucleic acid.

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5.1 Troubleshooting table, continued

NOTE: Refer to the FuGENE 6 web page for additional help:

https://www.wendangku.net/doc/0818264495.html,/techserv/fugene.htm

5.2 How to contact Roche Molecular Biochemicals

Three ways to contact us To contact Roche Molecular Biochemicals for technical assistance, choose one of the following:

Problem Possible cause Recommendation

Signs of cytotoxicity (continued)Plasmid prepara-

tion contami-

nated with large

amounts of endo-

toxin

Endotoxin is reported to be cytotoxic to some very sensi-

tive cell lines (e.g., Huh-7) and primary cultures (13). By

using FuGENE 6 Reagent for many common cell types it

may be possible to use DNA containing higher endotoxin

levels. See section 4.2.

If above tests

prove negative,

FuGENE 6

Reagent may be

cytotoxic to your

specific cell type

If you are using a very sensitive cell line, some steps can

be taken to minimize cytotoxicity:

?Perform the transfection in the presence of FBS.

?Reduce the time of exposure to the transfection

reagent:DNA complex, 2–3 h maximum, then replace

the medium.

?Perform the transfection at a higher cell density.

?Use different ratios of FuGENE 6 Reagent:DNA

If you are using…THEN…

the Internet access our web site at https://www.wendangku.net/doc/0818264495.html,

E-mail Identify the address that corresponds to your particular

location on the back cover of this instruction manual.

the telephone Identify the address that corresponds to your particular

location on the back cover of this instruction manual.

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5.3 References

General transfection techniques

1.Graham, R.L. and van der Erb, A.J.A. (1973) Virology52: 456.

2.Andreason, G.L. and Evans, G.A. (1988) BioTechniques6(7): 650.

3.Shigikawa, K. and Dover, W.J. (1988) BioTechniques6: 742.

4.Perkus, M.E. et al. (1993) J. Tiss. Cult. Meth.15: 72.

5.Keown, W., Campbell, C. and Kucherlapati, R. (1990) Methods Enzymol. 185:527.

6.Sambrook, J, Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning,

A Laboratory Manual; Volume 3, Second edition. Cold Spring Harbor Laboratory.

7.Kriegler, M. (1990) Gene Transfer and Expression: A Laboratory Manual, Stockton

Press, New York.

8.Murray, E.J., editor (1990) Gene Transfer and Expression Protocols: Methods in

Molecular Biology, Volume 7, Humana Press, Clifton, New Jersey.

9.Ausubel, F.M., Brent, R., Kingston, R.E., Moor, D.D., Seidman, J.G., Smith, J.A. and

Shuhl, K., editors (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc.

10.Felgner, J. et. al. (1993) J. Tiss. Cult. Meth. 15: 63.

11.Remy, J-S., Sirlin, C., Vierling, P. and Behr, J-P. (1994) Bioconjugate Chem.5: 647-654.

12.Haensler, J. and Szoka, F.C., Jr. (1993) Bioconjugate Chem.4: 372-379.

Endotoxin levels in primary cultures

13.Cotten, M., Baker, A., Saltik, M., Wagner, E. and Buschle, M. (1994) Gene Therapy1:

239-246.

Roche Molecular Biochemicals

5.4 Related products

continued on next page

Transfection Reagents

Cat. No.Pack Size DOSPER Liposomal Transfection Reagent 1 781 9951 811 169 2 ml (5 x 0.4 ml)

0.4 ml

DOTAP Liposomal Transfection Reagent

1 20

2 3751 811 177 2 ml (5 x 0.4 ml)

0.4 ml

Selection Antibiotics Cat. No.Pack Size Geneticin (G 418) 1 464 9731 464 9811 464 990250 mg

1 g 5 g Hygromycin B

843 555 1 g

Reporter Gene Assays Cat. No.Pack Size β-Gal ELISA

1 539 426 1 kit (19

2 tests)

β-Gal Reporter Gene Assay, chemiluminescent

1 758 241

1 kit

(500 assays, MTP format;250 assays, tube format)

β-Gal Staining Set 1 828 673 1 set (100 tests)CAT ELISA 1 363 727 1 kit (192 tests)CAT Staining Set 1 836 358 1 set (100 tests with

3.5 cm dishes)

Complete Protease Inhibitor Cocktail Tablets 1 697 49820 tablets hGH ELISA 1 585 878 1 kit (192 tests)Luciferase Reporter Gene Assay, high light intensity 1 669 8931 814 036200 assays

1000 assays

Luciferase Reporter Gene Assay, constant light signal 1 897 6671000 assays SEAP Reporter Gene Assay, chemiluminescent 1 779 842 1 kit

(500 assays, MTP format; 250 assays, tube format)

X-Gal 651 745651 737100 081745 740250 mg

25 mg 100 mg 1 g Mycoplasma Detection and Elimination

Cat. No.Pack Size BM Cyclin

799 05037.5 mg

(for 2 x 2.5 l medium)

DAPI (fluorescent detection of mycoplasma)236 27610 mg

Mycoplasma Detection Kit 1 296 744 1 kit (25 tests)Mycoplasma PCR ELISA

1 663 925

1 kit (96 reactions)

1814443.FM Page 18 Tuesday, August 21, 2001 10:58 AM

Roche Molecular Biochemicals

5.4 Related products , continued

Intelli-Search, Complete, pHB6, pVB6, pXB, pBH, pBV, pBX, pHM6, pVM6, pXM, pMH, pMV, and pMX are trademarks of a member of the Roche Group.FuGENE ? is a trademark of Fugent, L.L.C.

Geneticin is a registered trademark of Life Technologies, Inc.

Vectors and Screening Kits

Cat. No.Pack Size

pXB Bacterial Expression Vector (N-terminal) 1 814 59120 μg pBX Bacterial Expression Vector (C-terminal) 1 814 62120 μg pXM Mammalian Expression Vector (N-terminal) 1 814 69920 μg pMX Mammalian Expression Vector (C-terminal) 1 814 73720 μg pHB6 Bacterial Expression Vector (N-HA+His 6-C) 1 814 57520 μg pVB6 Bacterial Expression Vector (N-VSV-G+His 6-C)

1 814 58320 μg

pBH Bacterial Expression Vector (HA-C)

1 814 60520 μg pBV Bacterial Expression Vector (N-VSV-G-C) 1 814 61320 μg pVM6 Mammalian Expression Vector (N-VSV G+His 6-C)

1 814 67220 μg pHM6 Expression Vector (N-terminal HA tag/C-terminal HIS-6 tag, and β-Gal control vector) 1 814 66420 mg

pMH Mammalian Expression Vector (HA -C) 1 814 70220 μg pMV Mammalian Expression Vector (VSV-G-C) 1 814 72920 μg

Intelli-Search B Bacterial Colony Screen

(for identification of inserts in bacterial expression vectors)

1 814 842100 reactions Intelli-Search M Bacterial Colony Screen

(for identification of inserts in mammalian expression vectors)

1 814 834

100 reactions

1814443.FM Page 19 Tuesday, August 21, 2001 10:58 AM

1814443.FM Page 20 Tuesday, August 21, 2001 10:58 AM

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LIPOFECTAMINE 2000转染试剂转染步骤

LIPOFECTAMINE 2000转染试剂转染步骤 24孔板贴壁细胞的瞬时或稳定转染实验步骤：（在生长培养基中直接加入复合物） 1.转染前一天，胰酶消化细胞并计数，将细胞转至24孔板，控制密度使其在转染日密度接近90%。细胞铺板在0.5ml含血清，不含抗生素的正常生长的培养基中。 2.对于每孔细胞，使用50μl OPTI-MEMⅠ培养基稀释1μl-3μl LIPOFECTAMINE 2000试剂。温柔混匀LIPOFECTAMINE 2000，室温温浴5分钟（在5-25分钟内同稀释的DNA混合。保温时间过长会降低活性。可以批量制备。）注意：即使LIPOFECTAMINE 2000使用OPTI-MEMⅠ稀释，细胞也可以使用D-MEM培养。 3.对于每孔细胞，使用50μl无血清培养基（如OPTI-MEMⅠ培养基）稀释0.8μg-1.0μg DNA。多孔操作可以批量制备。 4.混合稀释的DNA（由第3步）和稀释的LIPOFECTAMINE 2000（由第2步）。在室温保温20分钟。注意：溶液可能会混浊，但不会影响转染。复合物可以在室温保持6小时稳定。

5.直接将复合物（100μl）加入到每孔中，前后（或左右）摇动培养板，轻轻混匀。注意：如果在无血清条件下转染，使用含血清的正常生长培养基进行细胞铺板。在加入复合物前移去生长培养基，替换为0.2ml无血清培养基。 6.在37℃，5％的CO2中保温18-48小时，无须去掉复合物或更换培养基或者在4-5小时后更换生长培养基也不会降低转染活性。 7.在细胞中加入复合物18-72小时后，分析细胞抽提物或进行原位细胞染色，检测报告基因活性。这依赖于细胞类型和启动子活性。对稳定表达，在开始转染一天后将细胞传代至新鲜培养基中（1：10），两天后加入筛选抗生素。进行稳定表达需要数天或数周。

细胞转染经验

转染注意因素有血清时的转染血清一度曾被认为会降低转染效率，但只要在DNA-阳离子脂质体复合物形成时不含血清，在转染过程中是可以使用血清的。转染过程在两步中需要使用培养基做为稀释液：在DNA-阳离子脂质体复合物准备过程以及复合物同细胞接触过程。在开始准备DNA和阳离子脂质体试剂稀释液时要使用无血清的培养基，因为血清会影响复合物的形成。但在复合物形成后，在加入细胞中前可以加入血清。阳离子脂质体和DNA 的最佳量在使用血清时会有所不同，因此如果你想在转染培养基中加入血清需要对条件进行优化。大部分细胞可以在无血清培养基中几个小时内保持健康。对于对血清缺乏比较敏感的细胞，可以使用 OPTI-MEMⅠ培养基，一种营养丰富的无血清培养基，或者在转染培养基中使用血清。对于对血清缺乏比较敏感的贴壁细胞，建议使用LIPOFECTAMINE 2000。培养基中的抗生素抗生素，比如青霉素和链霉素，是影响转染的培养基添加物。这些抗生素一般对于真核细胞无毒，但阳离子脂质体试剂增加了细胞的通透性，使抗生素可以进入细胞。这降低了细胞的活性，导致转染效率低。所以，在转染培养基中不能使用抗生素，甚至在准备转染前进行细胞铺板时也要避免使用抗生素。这样，在转染前也不必润洗细胞。对于稳定转染，不要在选择性培养基中使用青霉素和链霉素，因为这些抗生素是GENETICIN选择性抗生素的竞争性抑制剂。另外，为了保证无血清培养基中细胞的健康生长，使用比含血清培养基更少的抗生素量。细胞维护和培养的演变可以通过常规的次培养步骤保持转染铺板前的细胞健康。每周传代一到两次，稀释程度使得下次传代前细胞几乎融合。不要使细胞保持融合超过24小时。大多数已建立的细胞系都是非整倍体，原代培养包括了表达不同基因组合的细胞的混合物。细胞培养在实验室中保存数月和数年后会经历突变，总染色体重组或基因调控变化等而演化。这会导致和转染相关的细胞行为的变化。如果随时间发现这种变化，融化一管新鲜的细胞可能会恢复原先的转染活性。比如，新鲜融化的NIH 3T3细胞比传代8次的细胞表现出更高的转染效率（图13）。融化细胞的进一步传代并没有降低转染效率。因此，如果观察到转染效率降低，可以试着转染新鲜培养的细胞以恢复最佳结果。或者，几种来源于经筛选，转染效率较高细胞亚系的细胞系现在有售。细胞铺板密度用于转染的最佳细胞密度根据不同的细胞类型或应用而异。一般转染时，贴壁细胞密度为70%-90%，悬浮细胞密度为2×106-4×106细胞/ml时效果较好。确保转染时细胞没有长满或处于静止期。因为转染效率对细胞密度很敏感，所以在不同实验间保持一个基本的传代步骤很重要。铺板细胞数目的增加可以增加转染活性和细胞产量。在三种不同密度进行细胞铺板的比较表明铺板密度最高的，CAT活性也最高（图14）。得到最高活性所需的LIPOFECTAMINE试剂的量也相应增加了。这些结果说明，对于转染相同量的DNA所需的最佳阳离子脂质体试剂的量会因细胞密度而异。

各种转染试剂的中文转染方法

各种转染试剂的中文转染方法 FuGENE6（Roche）转染步骤：转染前一天将细胞分至培养板，转染当天细胞应50-80％融合。将细胞以1-3×105/2 ml接种于6孔板后孵育过夜将达到如此密度。将FuGENE6 Reagent在室温孵育10-15分钟。使用之前将FuGENE6颠倒混匀一下。 1. 在PCR管中加入不含血清和双抗的营养液以稀释FuGENE6，直至总体积到100 ul。 2. 将3-6 ul FuGENE6 Reagent直接加入营养液，轻弹管壁混合。 3. 加入1-2 ug的DNA溶液（0.02－2.0 ug/ul），轻弹管壁混合。 4. 室温孵育20分钟。 5. 将6孔板中的旧营养液吸出，加入约1 ml不含血清和双抗的营养液洗涤一次，再加入2 ml不含血清和双抗的营养液。 6. 将转染复合物加入细胞，混匀使之均匀分布。 7. 3-8小时后，加入血清或换成含血清的营养液。 Lipofectamine 2000(Invitrogen)转染试剂转染步骤（6孔板）： 1. 转染前一天，胰酶消化细胞并计数，细胞铺板，使其在转染日密度为90-95％。细胞铺板在2 ml含血清，不含抗生素的正常生长的培养基中。 2. 对于每孔细胞，使用250 ul无血清培养基（如OPTI-MEM I培养基）稀释4.0 ugDNA，轻轻混匀。 3. 使用前将Lipofectamine 2000转染试剂轻轻混匀，用250 ul无血清培养基（如OPTI-MEM I培养基）稀释10 ul Lipofectamine 2000转染试剂，轻轻混匀。Lipofectamine 2000稀释后，在5分钟内同稀释的DNA混合（<30分钟）。NOTE：若使用DMEM培养基，则需在5分钟内同稀释的DNA混合。 4. 混合稀释的DNA（第二步）和稀释的Lipofectamine 2000（第三步）。室温放置20分钟。 5. （optional）将6孔板中的旧营养液吸出，用无血清培养基清洗两次。加入2 ml无血清配养基。 6. 直接将复合物加入到每孔中，摇动培养板，轻轻混匀。中保温24-48小时。无需去掉复合物或更换培养基。 7. 在37℃，5％CO 2 或者在4-5小时后更换培养生长基也不会降低转染活性。 8. 在细胞中加入复合物24-72小时后，分析细胞抽提物或进行原位细胞染色，检测报告基因活性。这依赖于细胞类型和启动子活性。对稳定表达，在开始转染一天后将细胞传代至新鲜培养基中，两天后加入筛选抗生素。进行稳定表达需要数天或数周。贴壁细胞的稳定转染：转染后24小时，将细胞以≥1:10的比例传代至新鲜培养基中，次日加入选择性培养基。 Lipofectamine 2000转染试剂转染步骤（24孔板）：

磷酸钙法细胞转染试剂盒

磷酸钙法细胞转染试剂盒简介：外源基因导入真核细胞的方法有很多种，如磷酸钙转染法、DEAE-葡聚糖转染法、脂质体法、电穿孔法、显微注射法等。Leagene 磷酸钙法细胞转染试剂盒(Calcium Phosphate Cell Transfection Kit)是在传统的磷酸钙细胞转染方法的基础上进行了改良，提高了转染效率，并降低了毒性，可用于磷酸钙法转染细胞，不仅可以瞬时表达，也可以筛选稳定株。组成：操作步骤(仅供参考)： (一)贴壁细胞转染： 1、在转染前24h 用胰蛋白酶消化培养细胞，取适量对数期细胞转移至新的培养器皿中，待细胞密度大70～80%满时即可进行转染。后续操作步骤均按6孔板计算，如果转染器皿不同，请按比例自行调节用量。 2、在加入DNA 之前2～4h ，加入2ml 不含抗生素的完全培养液，置于37℃ 5% CO 2培养箱培养。 3、取DNA(体积不宜超过20μl)加入100μl Calcium chloride solution ，混匀，即为DNA-CaCl 2溶液。 4、取BBS solution 100μl ，用移液器一边吹打BBS solution ，一边逐滴加入DNA-CaCl 2溶液(操作缓慢，一般在1～2min)。 5、室温静置20～30min ，即为DNA-CaCl 2-BBS 溶液，此时可能出现极其微小颗粒沉淀。 6、取DNA-CaCl 2-BBS 溶液底部物质均匀加入到6孔板细胞中，轻轻晃动混匀。 7、置于37℃ 5% CO 2培养箱培养。 8、去除培养液，用PBS 清洗细胞2次，加入2ml 完全培养液继续培养，一般24h 后可见转染细胞的表达。 (二)悬浮细胞转染： 1、低速离心收集悬浮细胞，用PBS 洗涤1次。 2、取DNA(体积不宜超过20μl)加Calcium chloride solution ，混匀，即为DNA-CaCl 2溶液。编号名称 CZ0008 100T CZ0008 200T Storage 试剂(A): Calcium chloride solution 10ml 20ml -20℃ 试剂(B): BBS solution 10ml 20ml -20℃ 使用说明书 1份

转染步骤及经验(精华)

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jetPRIME转染试剂说明

Polyplus-transfection S.A. - Bioparc - 850 Bd S. Brant - 67400 Illkirch - France - Phone: +33 3 90 40 61 80 - Fax: +33 3 90 40 61 81 Polyplus-transfection Inc. - 1251 Ave of the Americas - 34th fl. - New-York - NY 10020 - USA https://www.wendangku.net/doc/0818264495.html, jetPRIME? in vitro DNA & siRNA transfection reagent PROTOCOL DESCRIPTION jetPRIME? is a novel powerful molecule based on a polymer formulation manufactured at Polyplus-transfection?. jetPRIME? ensures effective and reproducible DNA and siRNA transfection into mammalian cells. jetPRIME? is extremely efficient on a wide variety of cell lines. This powerful reagent only requires low amounts of nucleic acid per transfection, hence resulting in very low cytotoxicity . 1 Transient DNA transfection protocol (2) 1.1 Cell seeding .......................................................................................................................................... 2 1.2 DNA transfection protocol ................................................................................................................... 2 1.3 Virus production in adherent cells ....................................................................................................... 5 1.4 Optimization guidelines (5) 2 siRNA transfection protocol (6) 2.1 Cell seeding .......................................................................................................................................... 6 2.2 siRNA transfection protocol .. (7) 3 DNA & siRNA cotransfection protocol (7) 3.1 Cell seeding .......................................................................................................................................... 7 3.2 DNA & siRNA cotransfection protocol . (8) 4 Transfection of CRISPR/Cas9 ..................................................................................... 9 5 Stable DNA transfection ............................................................................................. 9 6 Troubleshooting ....................................................................................................... 10 7 Product information (11)

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转染试剂使用注意事项.doc

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细胞转染操作步骤

RNAi or siRNA Transfection 以24孔板为例，其余规格的转染见表1 1 中板，细胞密度为30-50%适宜。注意：根据转染后细胞检测时间长短决定细胞中板密度，如果转染后需要长时间后检测，则细胞中板密度适当降低，已避免细胞过度生长导致存活降低。 2 第二天（24-36小时后）每个孔转染方式如下： A 将20pmol siRNA溶于50ul Opti-mem无血清培养基中。 B 将1ul lipo2000溶于50ul Opti-mem无血清培养基中，混匀室温放置5min。 C 将A B两管混合，放置20min。 3 转染期间，将24孔板培养基换成无血清培养基，每孔400ul。将C管mix加入24孔板对应孔中，4-6小时候换成有血清培养基。 Plasmid DNA Transfection DNA(ug):lipo 2000(ul)=1:2-3 转染时细胞密度越高，转染效率，表达效率也越高，并且可以降低细胞毒性。 1 中板。贴壁细胞：0.5-2X105 cells/well，第二天待细胞密度达到90%以上时转染悬浮细胞：4-8X105 cells/well，中板后随即转染。 2 转染。 A 将0.8ug DNA溶于50ul Opti-mem无血清培养基中。 B 将2ul lipo2000溶于50ul Opti-mem无血清培养基中，混匀室温放置5min。 C 将A B两管混合，放置20min。转染期间，将24孔板培养基换成无血清培养基，每孔400ul。将C管mix

加入24孔板对应孔中，4-6小时候换成有血清培养基。 Table 1. Culture Shared reagents DNA transfection RNAi transfection 中板密度*Culture vessel Surf. area per well Vol. of plating medium Vol. of dilution medium DNA Lipofectamine ?2000 cell/well 96-well0.3cm2100ul2X25ul0.2ug0.5ul 0.5-2X105 cell/well 24-well2cm2500ul2X50ul0.8ug 2.0ul 1-3X105 cell/well 12-well4cm21ml2X100ul 1.6ug 4.0ul 2-3X105 cell/well 6-well (35mm) 10cm22ml2X250ul 4.0ug**10ul 8-10X105 cell/dish 60mm20cm24ml2X0.5ml8.0ug***20ul 2-3X106 cell/dish 10cm60cm215ml2X1.5ml24ug60ul *：中板密度根据不同细胞不同实验有所不同，这里仅提的数据仅供参

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脂质体转染的实验原理与操作步骤大全日期：2012-06-25 来源：互联网作者：青岚点击：3644次摘要：细胞转染的方法主要包括：电穿孔法、显微注射、基因枪、磷酸钙共沉淀法、脂质体转染法、多种阳离子物质介导、病毒介导的转染等，理想的细胞转染方法是具有高转染效率、对细胞的毒性作用小等，本文主要介绍细胞转染常用的方法-脂质体转染的原理和操作步骤等。找产品，上生物帮>> >> 细胞转染的方法主要包括：电穿孔法、显微注射、基因枪、磷酸钙共沉淀法、脂质体转染法、多种阳离子物质介导、病毒介导的转染等，理想的细胞转染方法是具有高转染效率、对细胞的毒性作用小等，本文主要介绍细胞转染常用的方法-脂质体转染的原理和操作步骤等。脂质体(lipofectin regeant，LR)试剂是阳离子脂质体N-[1-2，3-Dioleyoxy，Propyl]-n，n，n-Trimethylammonium Chloride(DOTMA)和Dioleoyl photidye-thanolamine(DOPE)的混合物[1：1(w/w)]。它适用于把DNA转染入悬浮或贴壁培养细胞中，是目前条件下最方便的转染方法之一。转染率高，优于磷酸钙法，比它高5~100倍，能把DNA和RNA转染到各种细胞。用LR进行转染时，首先需优化转染条件，应找出该批LR对转染某一特定细胞适合的用量、作用时间等，对每批LR都要做：第一，先要固定一个DNA的量和DNA/LR混合物与细胞相互作用的时间，DNA可从1~5μg和孵育时间6小时开始，按这两个参数绘出相应LR需用量的曲线，再选用LR和DNA两者最佳的剂量，确定出转染时间(2~24小时)。因LR对细胞有一定的毒性，转染时间以不超过24小时为宜。细胞种类：COS-7、BHK、NIH3T3、Hela和Jurkat等任何一种细胞均可作为受体细胞。一、脂质体(liposome)转染方法原理脂质体(liposome)转染方法原理：脂质体((Iiposome)作为体内和体外输送载体的工具，已经研究的十分广泛，用合成的阳离子脂类包裹DNA，同样可以通过融合而进人细胞。使用脂质体将DNA带人不同类型的真核细胞，与其它方法相比，有较高的效率和较好的重复性。中性脂质体是利用脂质膜包裹DNA，借助脂质膜将DNA导入细胞膜内。带正电的阳离子脂质体，DNA并没有预先包埋在脂质体中，而是带负电的DNA自动结合到带正电的脂质体上，形成DNA-阳离子脂质体复合物，从而吸附到带负电的细胞膜表面，经过内吞被导入细胞。二、脂质体转染操作步骤 1、操作步骤[方法一]：

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