We recommend 1:1 ratio of DNA and lipid. For example, 1.25 ug DNA and 1.25 ul of the reagent for one ml of suspension cells.

In general, the transfection efficiency for FreeStyleMAX reagent is 65% when used with suspension CHO and 293 cells.

1. Order of reagent addition was incorrect. Mix DNA with PLUS Reagent in dilution medium. Then combine this mixture with diluted LipofectAMINE Reagent.

2. Diluted DNA or PLUS Reagent was not mixed well before precomplexing. Be sure to mix DNA well when diluting before adding PLUS Reagent or vice versa. The order of addition of DNA and PLUS Reagent to the dilution medium for precomplexing has no effect on the transfection activity.

3. No LipofectAMINE Reagent was used. Ensure that DNA-PLUS Reagent complex is mixed and incubated with diluted LipofectAMINE Reagent before adding to cells.

Yes, the reagent can be used for adherent cells, however the protocol is an optimized protocol for CHO and 293 cells in suspension culture. If you want to transfect adherent cells please use Lipofectamine 2000 transfection reagent. Please use this link for the transfection protocol with Lipofectamine 2000.

http://www.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf

Lipofectamine 2000 is designed for adherent cells. FreeStyle 293fectin is designed for Hek 293 suspension cells. For transfecting 293 suspension cells, FreeStyle MAX reagent yields transfection efficiency comparable to Lipofectamine 2000 and 293fectin. However, Lipofectamine 2000 and 293fectin reagents do not work well for CHO suspension cells.

A small granular-like precipitate may be detected microscopically on the cells after transfection using cationic lipid. This is normal. The presence or absence of this precipitate is not indicative of the transfection efficiency.

The precipitate can be caused by presence of excess EDTA or cationic lipid. Use DNA in water or, if in TE, use EDTA concentrations of <0.3 mM in the diluted DNA. Ensure concentrations of cationic lipid reagents do not exceed recommended amounts in complex formation.

CELLFECTIN: Reagent 200ug/ml, DNA 20ug/ml.
DMRIE-C: Reagent 200ug/ml, DNA 20ug/ml.
LIPOFECTIN: Reagent 500ug/ml, DNA 100ug/ml.
LIPOFECTAMlNE™: Reagent 250ug/ml, DNA 10ug/ml.
LIPOFECTAMlNE™ 2000: Reagent 50ug/ml, DNA 50ug/ml.

DNA needs to be pure enough to be exposed to the cells otherwise they will lyse, or the transfection efficiency will be so low as to be ineffective. Both CsCl purification and column-based purification methods should produce high quality DNA. After any purification, be sure to remove all the ethanol before resuspending the DNA in sterile water or TE. It is highly recommended to do a "cells + DNA only" control to check for any adverse side effects of the DNA on the cells. Additionally, leave one plate with just cells and media as a control.
Column-based purification methods also yield DNA of suitable quality for transfection experiments. Please use PureLlink Hi Pure, PurLlink HQ and Charge Switch ER Plasmid Purification kits for transfection quality DNA isolation.