n-BLAST 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 protocol
Transfection of adherent cells
1.1. Hydrate n-Blast lipid film at room temperature with 0.75 ml (NB30075) or 1.5 ml (NB30150) of the hydration buffer. Vortex for 10 seconds at top speed before use. Store the hydrated reagent at 4 degree C and vortex briefly before each use.
1.2. Dilute the hydrated n-Blast reagent with serum-free medium.
1.3. Dilute the DNA with DNA diluent and incubate 1-5 minutes at room temperature. DO NOT INCUBATE for longer than 5 minutes and AVOID vortexing. Refer to Table 1 for the appropriate volume of serum-free medium.
Although n-Blast has been optimized for specific cell culture conditions, optimization may be needed to achieve maximum transfection ef iciency. The two critical variables are the ratio of n-Blast reagent to DNA and the quantity of DNA used. For optimization, first maintain a fixed ratio of n-Blast reagent to DNA, then vary the DNA quantity over the suggested range. If necessary, optimize the ratio of n-Blast reagent to DNA by using 3 to 6 ul of reagent for each 1 ug of DNA. Use a low DNA quantity to optimize this ratio. Following this process, cell number can also be optimized.
1.4. Add the DNA solution to the diluted n-Blast Transfection Reagent. Incubate at room temperature for 5 to 10 minutes to allow the n-Blast /DNA complexes to form.
Do not incubate DNA solution with the n-Blast 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.
Transfection of Suspension cells
For suspension cells, the protocol is the same as described for adherent cells, with the following exceptions:
The day before transfection, split the cells so they are in good condition on the day of transfection.
While the n-Blast/DNA complexes are incubating, spin down the cells, resuspend them at 1 x 106 or 2 x 106 cells/ml in medium with or without serum, and transfer the appropriate volume to the dish (Table 3).
Prepare the mixture of n-Blast/DNA complexes as above, add it directly to the cells, and mix well by gently pipetting 2 to 3 times. Incubate at 37 degree C and proceed as described for adherent cells.