CellPro™Plasmid Transfection Reagent HP (IC-6012)                         

BackGround

CellPro™Transfection Reagent which is a kind of new material transfection reagent, has low toxicity and high transfection efficiency, wide application range, product letter high price advantage is a reagent for the transfection of DNA and siRNA into eukaryotic cells. 

Storage 

Store at 4^oC. Note:Forbidden FREEZE.

Transfection Procedure 

A. Normal operation

1. Planting cells on the cell culture plate one day in advance, the cell confluence degree should be 60-80% at the time of transfection, and the cell state should be kept.

Transfection should be carried out in good condition.

2. The nucleic acid was directly mixed with the transfection reagent, and sufficiently mixed with the transfection reagent for about 15 times. After incubation at room temperature for 10 minutes, the nucleic acid-nanocomposite was prepared. During the preparation of the composite, no liquid residue was ensured on the tube wall.

3. The nucleic acid-nano-complex was added into the cells and blended gently, then put into the incubator for further culture.

4. After 24-48 hours of transfection, normal fluid exchange was performed on the cells.

B.Reverse operation

1.The nucleic acid was directly mixed with the transfection reagent, and sufficiently mixed with the transfection reagent for about 15 times. After incubation at room temperature for 10 minutes, the nucleic acid-nanocomposite was prepared. During the preparation of the composite, no liquid residue was ensured on the tube wall.

2. Cells in good condition with 60-80% convergence and nucleic acid-nanocomposites were added to the cell culture plate (regardless of the sequence) and gently blended, then placed in the incubator for further culture.

3. After 24-48 hours of transfection, normal fluid exchange was performed on the cells.

Important Guidelines

-The proportion of various nucleic acids was adjusted according to the following table and experimental requirements.

-Mix all kinds of nucleic acids before mixing with transfection reagents.

-If there is a small amount of precipitation before the use of the transfection reagent, please mix it with an oscillator without affecting the quality of the product.

-Plasmid DNA must be dissolved in ddH2O. If it is dissolved in Buffer, the transfection efficiency will decrease by 70%, even leading to the failure of transfection.

-Plasmid DNA must be de-endotoxin, otherwise the transfection efficiency will decrease by 70%, even lead to the failure of transfection.

-No reagent can be used to dilute the nucleic acid or transfection reagent in the preparation of the complex, just mix them proportionally. Otherwise, the transfection efficiency will decrease by 80%, even lead to the failure of transfection.

-After checking and mixing with transfection reagent, the pipette was used to blow and suck 15 times to mix sufficiently and ensure that there was no liquid residue in the tube wall.

-The nucleic acid was mixed with the transfection reagent and incubated at room temperature for at least 10 minutes.

-In the whole process of transfection experiment, the cells can be cultured in complete medium without using serum-free medium.

-If white or black round particles were observed under the microscope after transfection, they would be nanoparticles.

Optimizing  Transfection

1. The above data are all 24-hole single-hole plate as an example. For other specifications, 96-well plate divides the data from the above table by 4, 12-well plate multiplies the data from the above table by 2, and so on.

2. Gradient 1 is suitable for better transfected cells, such as HEK293T, Gradient 2 is suitable for relatively difficult transfected cells, such as RAW264.7, etc. For extremely difficult transfected cells, the actual amount of transfection is adjusted to 3ul-5ul according to the above table.

3. The ratio of plasmid DNA (ug) and siRNA (ul) to transfection reagent (ul) is 1:1, and the dosage can be designed according to this ratio.

4. The concentration of siRNA in the above table is 20 uM, if the concentration of siRNA is 40 uM, the amount of siRNA in the above table will be reduced by half; if the concentration is 10 uM, the amount of siRNA in the above table will be doubled, and so on.

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