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Question of transfection reagent

How can you improve low transfection efficiency with Lipofectin?

Incubate Lipofectin Reagent in OPTI-MEM I Medium (or other medium without serum) for 30 minutes prior to adding DNA to help improve the transfection efficiency upto 3-fold.

How crucial is the transfection efficiency when using the ViraPower Adenoviral System? Will low transfection efficiencies still prod

Getting a cell transfected and starting a productive viral infection are two different things. If only one or two cells in your lawn are producing virus, it will take quite a while for that to be visible to the naked eye - most people would give up before that happens. Transfection efficiency is correlated with the start of virus production simply in that the more cells you get DNA into, the more chance you have of seeing virus production within the first week or two. Lower transfection efficiencies will eventually produce virus but you have to wait a long time to see it.

Why is transfection not reproducible?

In general, efficiencies will show some degree of variability from transfection to transfection, no matter how hard one tries to control the parameters of transfection. Keep all transfection parameters, such as confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. To minimize variability from transfections, you can use internal reference control for example B galactosidase or luciferase. Cotransfect your expression plasmid with the reference plasmid and assay for the activity of B-gal/luciferase. See FOCUS 17.2, 60.

How does the use of LIPOFECTAMlNE 2000 reagent improve transfection over the other lipid reagents?

It generally yields better transfection activity (as measured by protein expression) than all other lipids in a majority of the cell lines tested. It includes a streamlined, simple protocol where the complexes are added directly to cells without changing media. This lends itself to high throughput applications. It works very well in the presence or absence of serum. Examples of cells that show the highest transfection efficiency with LIPOFECTAMlNE 2000 include 293 F, 293 H, BE(2)C, CHO-K1, CHO-S, COS-1, COS7-L, Human Primary Fibroblasts, Ht-29, HT-1080, MDCK, MRC-5, PC12 and Vero.

What transfection conditions do you recommend for the ViraPower Adenovirus Expression System?

We generally produce adenoviral stocks in 293A cells using the following optimized transfection conditions. The amount of adenovirus produced using these recommended conditions is approximately 10 ml of crude viral lysate with a titer ranging from 1 x 10e7 to 10e8 pfu/ml. We use Lipofectamine 2000 for transfection. If using another transfection reagent, use the manufacturer’s recommendations.

Tissue culture plate size: 6-well plate (one well per adenoviral construct) Number of 293A cells to transfect: 5 x 105 cells Amount of Pac I-digested pAd-DEST expression plasmid: 2 ug Amount of Lipofectamine 2000 6 ul

293A cells should be plated 24 hours prior to transfection in complete medium, and should be 90-95% confluent on the day of transfection. Make sure the cells are healthy at the time of plating.

What are the maximum lipid/DNA concentrations for preparing transfection complexes?

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.

Is it important to do a double CsCl purification of plasmid DNA prior to transfection? Is it possible to use column-based purification methods?

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.

Why do I have low/no transfection efficiency with LipofectAMINE PLUS reagent?

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.