Testing Data
n-Fect reagent
n-Fect
Synonyms
n-Fect; n-Fect reagent
Methods and Procedures
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.
To obtain maximum efficiency in particular cells, 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 of the DNA quantity used, maintain a fixed ratio of n-Fect reagent to DNA, and then vary the DNA quantity over a suggested range (see Table 2). See the Appendix for examples.
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. See the Appendix for examples.
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. Refer to Table 6 for the appropriate volume of 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. See the Appendix for examples.
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. Refer to Table 7 for suggested cell number according to culture dishes size and cell types. Refer to Table 8 for appropriate medium volumes.
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.
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.
To obtain maximum efficiency in particular cells, 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 of the DNA quantity used, maintain a fixed ratio of n-Fect reagent to DNA, and then vary the DNA quantity over a suggested range (see Table 2). See the Appendix for examples.
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. See the Appendix for examples.
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. Refer to Table 6 for the appropriate volume of 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. See the Appendix for examples.
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. Refer to Table 7 for suggested cell number according to culture dishes size and cell types. Refer to Table 8 for appropriate medium volumes.
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.
Preparation and Storage
For maximum stability store all reagents at 4 degree C upon receipt.
All components are stable for at least one year if stored properly.
All components are stable for at least one year if stored properly.
Testing Data
Testing Data
Related Product Information for n-Fect reagent
n-Fect Transfection Reagent is a novel cationic lipid specially formulated for optimal transfection in neuronal cells, including primary neurons, differentiated post-mitotic neurons, neuronal cell lines, and glial cells. n-Fect Transfection Reagent is compatible with serum eliminates the need to change media following transfection. An included DNA Diluent is designed to facilitate DNA/lipid complex (lipoplex) formation and enhance the transformation efficiency in certain neuronal cells such as NT2 (not recommended for primary and differentiated neurons). Cell type specific protocols are developed for nFect Transfection Reagents to ensure optimal transfection results.
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Product Notes
The n-Fect (Catalog #AAA555833) is a Reagent and is intended for research purposes only. The product is available for immediate purchase. It is sometimes possible for the material contained within the vial of "n-Fect, Reagent" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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