n-FECT Reagent

For maximum stability store all reagents at 4 degree C upon receipt.
All components are stable for at least one year if stored properly.

1. Transfection of Primary Neurons
1.1. Hydrate the n-Fect lipid vial at room temperature with 1.5 ml of the hydration buffer. Vortex for 30-60 seconds at top speed. Store the hydrated reagent at 4 degree C and vortex briefly before use.
1.2. Dilute the hydrated n-Fect reagent with serum-free medium. Refer to Table 1 for the appropriate volume of serum-free medium.
1.3. Dilute the DNA with the serum free medium (do not use the DNA Diluent for primary neurons). Refer to Table 1 for the appropriate volume of serum-free medium.
1.4. Add the DNA solution to the diluted n-Fect Transfection Reagent. Incubate at room temperature for 5 to 10 minutes to allow the n-Fect /DNA complexes to form.
Do not incubate DNA solution with the n-Fect Transfection Reagent for longer than 30 minutes
1.5. Add your complexes directly to the cells growing in serum-containing culture medium.
1.6. Add fresh growth media as needed 24 hours post transfection. Depending on the cell type and promoter activity, the assay for the reporter gene can be performed 24 to72 hours following transfection.
For some cell types, the old media can be replaced with fresh media at this step.
2. Transfection of Neuronal Cell Lines
2.1. Hydrate n-Fect lipid film at room temperature with 1.5 ml of the hydration buffer. Vortex for 3060 seconds at top speed. Store the hydrated reagent at 4 degree C and vortex briefly before use.
2.2. Dilute the hydrated n-Fect reagent with serum-free medium.
2.3. Dilute the DNA with the DNA Diluent and incubate 1 to 5 minutes at room temperature. Refer to Table 3 for the appropriate volume of DNA Diluent.
Do not incubate DNA with DNA Diluent for more than 5 min. Avoid vortexing the DNA Diluent.
Although n-Fect consistently delivers high transfection efficiencies, in order to obtain maximum efficiency in particular cell types, some optimization may be needed. The two critical variables are the ratio of n-Fect reagent to DNA and the quantity of DNA used. For optimization, first maintain a fixed ratio of n-Fect reagent to DNA, and then vary the DNA quantity over the suggested range. If necessary, optimize the ratio of n-Fect reagent to DNA by using 1.25 to 12.5
mul of reagent for each 1 mug of DNA. Use a low DNA quantity to optimize this ratio. Following this process, cell number can also be optimized.
2.4. Add the DNA solution to the diluted n-Fect Transfection Reagent. Incubate at room temperature for 5 to 10 minutes to allow the n-Fect /DNA complexes to form.
Do not incubate DNA solution with n-Fect Transfection Reagent for more than 30 minutes.
2.5 Add your complexes directly to the cells growing in serum-containing culture medium. Refer to Table 4 for suggested cell numbers for specific tissue culture dishes. Refer to Table 5 for appropriate medium volumes.
Cells plated the day before should be 50%-70% confluent on the day of transfection.
2.6 Add fresh growth media as needed 24 hours post transfection. Depending on the cell type and promoter activity, the assay for the reporter gene can be performed 24 to72 hours following transfection.
For some cell types, the old media can be replaced with fresh media at this step.
The same protocol can be used to produce stably transfected cells: 48 to 72 hours post transfection, put the cells in fresh medium containing the appropriate selection antibiotic. It is important to wait at least 48 hours before exposing the transfected cells to the selection media. For some cell types it may be necessary to wait as long as 4 to 5 days before applying the selection condition.
3. Transfection of Differentiated Post-Mitotic Neurons and Glial Cell Lines
3.1. Hydrate n-Fect lipid film at room temperature with 1.5 ml of the hydration buffer. Vortex for 3060 seconds at top speed. Store the hydrated reagent at 4 degree C and vortex briefly before use.
3.2. Dilute the hydrated n-Fect reagent with serum-free medium.
3.3. Dilute the DNA with the serum free medium. Refer to Table 6 for the appropriate volume of serum-free medium. Although n-Fect consistently delivers high transfection efficiencies, in order to obtain maximum efficiency in particular cell types, some optimization may be needed. The two critical variables are the ratio of n-Fect reagent to DNA and the quantity of DNA used. For optimization, first maintain a fixed ratio of n-Fect reagent to DNA, and then vary the DNA quantity over the suggested range. If necessary, optimize the ratio of n-Fect reagent to DNA by using 5 to 20 mul of reagent for each 1 mug of DNA. Use a low DNA quantity to optimize this ratio. Following this process, cell numbers can also be optimized.
3.4. Add the DNA solution to the diluted n-Fect Transfection Reagent. Incubate at room temperature for 5 to 10 minutes to allow the n-Fect /DNA complexes to form.
Do not incubate the DNA solution with the n-Fect Transfection Reagent for longer than 30 minutes
3.5. Add your complexes directly to the cells growing in serum-containing culture medium.
Cells plated the day before should be 50%-70% confluent on the day of transfection.
3.6. 24 hours post transfection, add fresh growth media as needed. Depending on the cell type and promoter activity, the assay for the reporter gene can be performed 24 to72 hours following transfection.
For some cell types, the old media can be replaced with fresh media at this step. Also, the same protocol can be used to produce stably transfected cells: 48 to 72 hours post transfection, put the cells in fresh medium containing the appropriate selection antibiotic. It is important to wait at least 48 hours before exposing the transfected cells to the selection media. For some cell types it may be necessary to wait as long as 4 to 5 days before applying the selection condition.