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DNA Transfection Reagent
Description
The Biotool DNA Transfection Reagent is a novel nano-material with
low cytotoxic formulations for efficient transfection of a broad range of
eukaryotic cells. Experimental uses include cellular analysis and
protein expression.
Components
Cat#: B35101
Cat#: B35102
DNA Transfection Reagent
1.5 ml
10 × 1.5 mL
Manual
1
1
Component
Storage
Biotool DNA Transfection Reagent should be stored at -20°C. There
is no detriment to repeated freezing and thawing on the performance
of Biotool DNA Transfection Reagent.
Notice
Optimization of experimental conditions may be necessary to obtain
the best results.
Protocol
Transfection Recommended Amounts
Component
96-well
24-well
12-well
6-well
DNA per well
0.2 µg
0.8 µg
1.6 µg
4.0 µg
Biotool Reagent per well
0.5 µL
2.0 µL
4.0 µL
10.0 µL
Use the following procedure to transfect DNA into mammalian cells in
a 24-well format. For other formats, see Scaling Up or Down
Transfections. All amounts and volumes are given on a per well basis.
Note: Optimization may be necessary.
1. Adherent cells: One day before transfection, plate 0.5-2×105 cells
in 500 µL of growth medium without antibiotics so that cells will be
60-80% confluent at the time of transfection.
Suspension cells: Just prior to preparing complexes, plate
4-8×105 cells in 500 µL of growth medium without antibiotics.
2. For each transfection sample, prepare complexes as follows:
a. Dilute DNA in 50 µL medium without serum. Mix gently.
b. Mix Biotool DNA Transfection Reagent gently before use, then
dilute the appropriate amount in 50 µL medium without serum.
Incubate for 5 minutes at room temperature.
Note: Proceed to Step c within 25 minutes.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: [email protected]
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: [email protected]
c. After the 5 minute incubation, combine the diluted DNA with
diluted Transfection Reagent (total volume = 100 µL). Mix gently
and incubate for 20 minutes at room temperature
(solution may appear cloudy).
Note: Complexes are stable for 6 hours at room temperature.
3. Add the 100 µL of complexes to each well containing cells and
medium. Mix gently by rocking the plate back and forth.
4. Incubate cells at 37°C in a CO2 incubator for 18-48 hours prior to
testing for transgene expression. Medium may be changed after
4-6 hours.
Troubleshooting
Problem
Low transfection
efficiency
Little DNA
Increasing the DNA to Biotool™ ratio
(2 to 3 µL Biotool™ per µg DNA)
Cell density
transfecting cells at 60% to 80%
confluency
Cells in culture
for a long time
(over 20 cell
passages)
Floating of DNA
Cell death
Suggestion
Possible reason
fragile cells
(e.g. primary
fibroblasts)
Using new fresh cells from liquid nitrogen
Gentle centrifugation of the culture
plate (5 min at 210 g)
- Change the medium 4 to 6 h after
transfection.
- Decrease the DNA amount.
- Decrease the volume of Biotool™
- Perform transfection in
serum-containing medium.
- Analyze the transfection efficiency 24 h
after transfection instead of 48 h
after transfection.
- Ensure that Biotool™ and DNA are
diluted into the serum free medium.
- Check that the plasmid is endotoxin free.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: [email protected]
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: [email protected]