2.

2.1

How to Use this Product

Before You Begin

2.3

Transfection Procedure

Allow X-tremeGENE HP DNA Transfection Reagent, DNA and diluent to equilibrate to +15 to +25°C. Briefly vortex the X-tremeGENE HP DNA Transfection Reagent vial.

Dilute DNA with appropriate diluent (e.g., serum-free

medium) to a final concentration of 1 µg plasmid DNA /100 µl medium (0.01 µg/µl). Mix gently.

Place 100 µl of diluent, containing 1 µg DNA into each of four sterile tubes labeled 1:1, 2:1, 3:1, and 4:1.

NUse a minimum of 100 µl of diluent. Lower volumes may

significantly decrease transfection efficiency.

LUse sterile tubes or tissue culture treated round-bottom,

96-well plates to produce the complex.Pipet the X-tremeGENE HP DNA Transfection Reagent (1, 2, 3, or 4 µl) directly into the medium containing the diluted DNA without coming into contact with the walls of the plastic tubes. Mix gently.

NTo avoid adversely affecting transfection efficiency, do not

allow undiluted X-tremeGENE HP DNA Transfection

Reagent to come into contact with plastic surfaces. Do not use siliconized pipette tips or tubes.Incubate the transfection reagent:DNA complex for 15 min-utes at +15 to +25°C.

LSome ratios and cell types may required longer incubation

(up to 30 min). Determine this for your particular cell line and the ratio used.Remove the culture vessel from the incubator. Removal of growth medium is not necessary. Add the transfection com-plex to the cells in a dropwise manner.

LSee Table 1 to determine component amounts corre-sponding to the surface area of the cell culture vessel used.

Gently shake or swirl the wells or flasks to ensure even distri-bution over the entire plate surface. If available, use a rotating platform shaker for 30 seconds at low speed for mixing 96-well plates.

Once the transfection reagent: DNA complex has been added to the cells, there is no need to replace with fresh medium (as may be necessary with other transfection reagents)

Required Amount of X-tremeGENE HP DNA Transfection Reagent

To optimize, first transfect a monolayer of cells that is 70 - 90% conflu-ent, using 1:1, 2:1, 3:1 and 4:1 ratios of microliter ( l) X-tremeGENE HP DNA Transfection Reagent to microgram ( g) DNA. A ratio of 3:1 of microliter ( l) X-tremeGENE HP DNA Transfection Reagent to micro-gram ( g) DNA has been shown to be optimal for many cell types.LLower cell confluencies have also been tested successfully.

The recommended starting concentration is a 3:1. For most cell types, these X-tremeGENE HP DNA Transfection Reagent to DNA ratios pro-vide excellent transfection efficiency.

LFurther optimization may increase transfection efficiency in your

particular application. In addition to varying the ratio, other param-eters may also be evaluated, such as the amount of transfection complex added. For additional optimization guidelines, see Section 3, Troubleshooting and visit www.51wendang.com DNA

For best results, accurately determine the plasmid DNA concentra-tion using 260-nm absorption; estimates of DNA by measuring gel band density are not recommended. Determine DNA purity using a 260 nm/280 nm ratio (the optimal ratio is 1.8).

Prepare the plasmid DNA solution using sterile TE (Tris/EDTA) buf-fer or sterile water at a concentration of 0.1 to 2.0 µg/µl.

Use high quality DNA preparation kits to obtain endotoxin-free DNA.

Cell Culture Conditions

Minimize intra- and inter-experimental variance in transfection effi-ciency using cells that are regularly passaged, proliferating well in a log-growth phase, and plated at a consistent density.

For best results, accurately quantify cell concentration using a hematocytometer or automated system.

Cells must be healthy and free of Mycoplasma.

Cells should have a low passage number to achieve best results.

Following transfection, incubate cells for 18 – 72 hours before Other Media Additives

measuring protein expression. The duration of incubation will In some cell types, antimicrobial agents (e.g., antibiotics and fungi-depend on many factors, including the transfected vector con-cides) commonly included in cell-culture media may adversely affect

struct, the cell type being transfected, the cell medium, cell the transfection efficiency of X-tremeGENE HP DNA Transfection

density, and the type of protein being expressed. After the Reagent. If possible, exclude additives in initial experiments. Once

incubation period, measure protein expression using an assay high-efficiency conditions have been established, these components

appropriate for your system.can be added back while monitoring transfection results. Cell growth

and/or transfection efficiency may be affected by variations in serum

Notes:quality and medium formulations.

LAs with any experiment, include appropriate controls. Prepare cul-Verification of Vector Functionture wells with cells that remain untransfected, cells with transfec-tion reagent alone, and cells with DNA alone.Optimize transfection conditions using a known positive-control

reporter gene construct before transfecting cells with a new vector LFor stable transfection experiments, the complex-containing construct:medium should be left unchanged until the cells are passaged. At

that time, include appropriate selection antibiotics (e.g., G 418 Determine transfection efficiency using a reporter gene assay, such

Solution or Hygromycin B).as -Gal*, Luciferase*, or SEAP*.

Sequence flanking vector insert regions to verify the integrity of LTo prepare transfection complexes for different-sized containers

or parallel experiments, adjust component amounts correspond-your new construct.

ing to the surface area of the cell culture vessel used (see Table 1).2.2Preparation of Cells for Transfection

Adherent Cells: Plate cells approximately 24 hours before transfec-LFor ease-of-use when transfecting small volumes into 96-well

tion making sure cells are at the optimal concentration in the appropri-plates containing 0.1 ml culture medium per well, prepare 100 µl

ate cell culture vessel.of transfection complex, and then add 10 µl to each well (depend-ing on cell type).Suspension Cells: Plate freshly passaged cells at optimal concentra-tion.LThe optimal ratio of transfection reagent to DNA, and the optimal

total amount of complex, will depend on the cell line, cell density, day of assay, and gene expressed.

LAfter performing the optimization experiment in which several dif-ferent ratios are tested, select a ratio in the middle of the plateau optimum for future experiments.