High Efficiency Transfection (HET) Kit
Technical Manual: High Efficiency Transfection (HET) Kit
Cat. No: BW11002
Components: Buffer A: 50 ml; Buffer B: 5 ml
Product Description
The High Efficiency Transfection Kit (HET) developed by Biowit is a modified calcium phosphate transfection reagent and can be used to efficiently transfect many proliferative mammalian cells without causing cytotoxicity.
Standard Transfection Procedure
The optimal cell confluency for transfection is no more than 80%. The optimal seeding density may vary from different types of cell lines or primary cells and depends upon cell doubling time. Generally, cells are seeded at a density of 3～5× 106 cells/100-mm dish, 1～2×106 cells/60- mm dish or 6～8×105 cells/well for 6-well plates. In most cases, the transfection efficiency usually increases as more amount of plasmid DNA is added, while the DNA amount had better be within the range as indicated in the Table below.
1) The components are incubated at room temperature before use. cells are seeded into 100-mm dishes, 60-mm dishes or 6-well plates at appropriate density and incubated overnight at 37°C in a humidified CO2 incubator. Next day, replace with fresh media (without penicilin-streptomycin or withpenicilin-streptomycin) into the dishes or plates.
2) Prepare two sterile tubes labeled as A and B, respectively.
|
For a 100-mm dish
containing 5ml of medium: |
For a 60-mm dish
containing 2.5ml of medium: |
For one well of a 6-well plate containing 1ml of medium: |
|
Ttube A:
500μl Buffer A |
Tube A:
250μl Buffer A |
Tube A:
50μl Buffer A |
|
Tube B:
50μl Buffer B
10~100μg DNA
Appropriate volume of sterile H2O:
a final volume of 500μl |
Tube B:
25μl Buffer B
5～50μg DNA
Appropriate volume of sterile H2O:
a final volume of 250μl |
Tube B:
5μl Buffer B
1～10μg DNA
Appropriate volume of sterile H2O:
a final volume of 50μl |
3) Using a Pasteur pipette, mix and add solution B dropwise to solution A while bubbling air through solution A with another pipette. This step should be done within 2 minutes. And then incubate at room temperature for 30 minutes.
4) Add the mixture dropwise to the culture, and then gently swirl dishes or plates .
5) Incubate overnight at 37°C in a humidified CO2 incubator.
6) Replace with fresh medium and incubate at 37°C in a humidified CO2 incubator for 24～48h.
7) Check the transfection efficiency by available methods (e.g., immunofluorescence, western blot).
Storage
The kit can be stored for 3 months at room temperature, for 1 year at 4℃ and over 1 year at -20℃.
Usage
The product is for research only and may not be allowed for usage as drugs, agricultural or pesticidal products, food additives or household chemicals.
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