transfection reagent
 
 
Can Lipofectamine LTX be used in transfection of Suspension cells and other hard to transfect cells?

We have tested transfection of suspension cells such as Jurkat and K562. We achieved transfection efficiency of less than 5% for each of them. We do not notice toxicity with Lipofectamine LTX reagent when used with these suspension cells. For other cell lines please follow the guidelines in the product insert. However if you experience low transfection efficiency, then alternate methods such as electroporation/viral delivery methods are recommended. Please use the following link for cell specific protocols for K562 and Jurkat cells.


 

How does Lipofectamine LTX compare to Lipofectamine 2000?

Lipofectamine LTX and Lipofectamine 2000 are both recommended for plasmid DNA transfection. However, in a panel of 16 cell lines tested, LTX showed an average of 2.5 fold increase in expression and 25% lower cytotoxicity compared to Lipofectamine 2000.


 

The protocol sent out with Lipofectamine 2000 indicates to use 24 well plates but I am interested in using 60mm plates. How do I scale up my transfection with lipofectamine 2000?

A 60mm plate is 10x greater in surface area than a well from a 24-well plate. Therefore, you would need to scale up all of the components in the reaction by 10x.

The product insert for Lipofectamine 2000 (included with the reagent or can be downloaded from the web site) contains a table indicating the relative surface areas of different cell culture vessels and what factor needed to scale your transfections.


 

Is Lipofectamine 2000 the same as Lipofectamine?

No. These are two different formulations of lipids and each have been shown to transfect at different efficiencies different cell lines. Please see the Cell Line Database in order to see the optimal usage of either with your cell line.


 

Is Lipofectamine 2000 stable at room temperature?

Here is information we have regarding stability for this reagent:
LipofectAmine 2000 should be stored at 4oC. It is stable for at least one year.
It is stable for at least 6 months at room temperature if unopened.
It should survive at least one freeze-thaw.
DNA-LF2000 Reagent complexes are stable for at least 6 h at room temperature.


 

How to improve transfection efficiency with cationic lipids?

1. Select the cationic lipid reagent likely to result in highest transfection efficiency for your cell type. See the references listed in the Transfection Collection on our web site.

2. Optimize cationic lipid reagent and DNA amounts. The most important parameter after the condition of the cells is the ratio of lipid to DNA.

3. Do not use serum during complex formation. Serum contains lipids which will interfere with complex formation.OPTI-MEM I Reduced-Serun Medium or DMEM (use either without adding serum during complex formation) is good media for complex formation.

4. Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare DNA-cationic lipid reagent complexes.

5. Cell density should be 50% to 80% confluent at the time of transfection (for Lipofectamine 2000, we recommend >90% confluency). Cells should be in the mid-log growth phase.

6.Make sure the promoter-enhancer of the transfected DNA is compatible with the target cell type.

7.Do not use cationic lipid reagent that has been frozen or stored in a section of the refrigerator where the temperature is <4 degree.

8.Include a positive control for the transfection assay.


 

How can I optimize transfection conditions for Lipofectamine 2000?

 Optimization of transfection conditions is essential for the highest-efficiency transfections and the lowest toxicity. The conditions that should be optimized include Lipofectamine 2000 and DNA concentrations, and cell number. It is recommended that conditions be tested and optimized for cell lines, which do not have cell specific protocols available.

To optimize the amount of Lipofectamine 2000 reagent, start with cells at >90% confluency and 0.8-1.2 ยตg DNA for 24-well plates. With cell number and DNA concentration held constant, vary the amount of Lipofectamine 2000 Reagent to determine the optimal concentration (usually 1.5-3 ยตl). The cell number and amount of DNA can also be optimized. It is possible to minimize the effect of transfection on cell growth and viability by increasing the number of cells plated per well or by decreasing either Lipofectamine 2000 reagent or DNA concentrations. With careful optimization, this can be achieved with little impact on the level of transgene expression. 


 

Why are most cells dead after transfection using cationic lipid?

1. Too much DNA or lipid used. Do a dose-response curve to determine the optimal amount of DNA and cationic lipid reagent. DNA or cationic lipid alone generally has a minimal effect on cell growth. Consult the technical manual for optimization protocols for a specific lipid.

2. Do not use chloroquine, penicillin, or streptomycin during transfection because cationic lipid reagents make cells more sensitive.

3. Do a dose-response curve to determine the optimal number of cells/transfection.

4. Use OPTI-MEM I Medium during transfection. Reduce or omit the number of washes in serum-free medium. Use 5% to 10% serum in the transfection medium. Be sure to form complexes in the absence of serum.

5. Do not vortex or agitate cationic lipid reagents excessively; this may form cationic lipid reagent peroxides. 6.For stable transfection, allow at least 48 h for cells to express resistance before adding selective antibiotic.


 

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.


 

Can FreeStyle MAX reagent be used for transfecting adherent 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


 

How does FreeStyle MAX reagent compare to Lipofectamine 2000 and 293fectin?

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.


 

Why do I see precipitate on the cells after transfection?

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.


 

What is the recommended ratio of DNA and transfection reagent when transfecting

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.


 

What is the recommended ratio of DNA and transfection reagent when transfecting

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


 

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