PHP试剂,转染试剂

Clarkfection 是阳离子聚合物类型的转染试剂，相比脂质体类型具有更小的细胞毒性、更高的转染效率 以及更高的可重复性。

Clarkfection® is a superior cationic polymer-based transfection reagent. Compared with liposomes on the market, Clarkfection exhibits lower cytotoxicity, higher transfection efficiency and higher repeatability.

Clarkfection Features

For eukaryotic cells like HEK 293;

For both adherent and suspension cells;

For operations with or without serum;

More than 200,000 L application.

High protein expression and low cytotoxicity(EGFP protein expression transfected Clarkfection )

293E

293FT

COS7

HepG2

BHK21

MCF7

SW480

CHO-DG44

Clarkfection Transfection Protocol

The transfection procedures of adherent cells are shown in Fig. 1.

we take the 96-well plates as example：

4cells/well (table 1)and cultured with 5%CO2 at 37℃ for 18-24h before transfection.

2. Preparation of transfection mixture:

(1). Dilute DNA into serum-free medium(25μL in total) and homogenize gently.

(2). Dilute Clarkfection into serum-free medium(25μL in total) and homogenize gently and incubate at room temperature for 5 min.

(3). Mix the DNA and Clarkfection at room temperature for 15~20min 3. Remove the culture medium and add 50μL mixture per well.

4. After 4-6hours(for SF9 cell is 2h), remove the transfection mixture and add medium with serum.

5. Gene expression is tested after incubation with 5% CO2 at 37℃ for 48-72h(incubate sf9 cell line at 27℃ for 48-72h).

Fig. 1 Adherent cells' Transfection

Attention:

1). The experiment conditions for different cell lines are shown in Table 1. The corresponding transfection protocols can be got by entering links in Table 1.

(2). Number of cells per well, dosage of Clarkfection, DNA and serum-free medium for dilution are proportional to basal area per well for plates of different sizes. The basical areas for plates with different sizes are shown in Table 2. The dosage and ratio of DNA and Clarkfection should be optimized to achieve the best transfection results.

Table1. Usage of Clarkfection in different cell lines(96-well plate)

Cell type

Culture medium

Cells per well

DNA

Clarkfection

Medium change after 4-6h

DMEM

3×104

0.2µg

0.5µL

DMEM+10%FBS

DMEM

3×104

0.2µg

0.5µL

DMEM+10%FBS

DMEM

3×104

0.2µg

0.5µL

DMEM+10%FBS

DMEM

3×104

0.2µg

0.5µL

DMEM+10%FBS

DMEM

1.5×104

0.4µg

0.5µL

DMEM+10%FBS

DMEM

2×104

0.3µg

0.5µL

1640+15%FBS

MEM

3.5×104

0.3µg

0.75µL

MEM+10%FBS

MEM

2×104

0.2µg

0.5µL

MEM+10%FBS

DMEM+HT+pro

2×104

0.5µg

0.5µL

DMEM+HT+pro +10%FBS

DMEM

3×104

0.2µg

0.5µL

DMEM +10%FBS

MEM/NEAA+0.01mg/mL insulin + sodium pyruvat

2×104

0.1µg

0.25µL

MEM/NEAA+0.01mg/mL insulin + sodium pyruvat+10%FBS

IMDM

3×104

0.4µg

0.5µL

IMDM +10%FBS

DMEM

4×104

0.6µg

1µL

DMEM+10%FBS

IMDM+Pro

3×104

0.2µg

0.5µL

IMDM+Pro +10%FBS

DMEM

3×104

0.5µg

0.75µL

DMEM+10%FBS

DMEM

2×104

0.3µg

0.5µL

DMEM+10%FBS

DMEM

1.5×104

0.1µg

0.75µL

DMEM+10%FBS

DMEM

3×104

0.3µg

0.75µL

DMEM+10%FBS

SIM SF

5×104

0.4µg

0.75µL

SIM SF+10%FBS

Table 2. Scaling up or down transfections with Clarkfection(according to the growth area).

Note: the actual conditions should be optimized by experiments.