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General

Freezing can alter the integrity and composition of the lipid particles. The performance of the reagent may be affected, and it may only work for the first week or so. It is safer to purchase a new vial to ensure function. 

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Here are some reasons why you may be getting low transfection efficiency, along with suggested solutions. 

Here are possible causes for reduced viability following transfection, along with suggested solutions. Please note that as per recent findings, antibiotics can be used in media during transfection. We have compared transfecting cells in media with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.

Please review these possible causes for non-reproducible transfections, along with suggested solutions. Also, we recommend setting up one master mix of the DNA/lipid complex for the number of transfections planned, to reduce multiple pipetting errors. When adding your complexes, we recommend changing tips between wells since re-used tips could bring carryover, especially for the 96- or 384-well format with small-volume formats.

A common reason for seeing precipitate on your cells following lipid-based transfection is if there is excess EDTA or cationic lipid present. We recommend diluting the DNA in water or, if TE is preferred, use EDTA concentrations of <0.3 mM. Also ensure that concentrations of cationic lipid reagents do not exceed recommended amounts during complex formation. The presence or absence of this precipitate is not indicative of the transfection performance. 

Transfection with cationic lipids can produce light granular orange background fluorescence. The orange fluorescence is associated with the lipid/DNA complexes and is not related to GFP. This background varies depending on the cationic lipid reagent used and does not interfere with transfection results.  

Lipofectamine® 3000 Transfection Reagent

Freezing can alter the integrity and composition of the lipid particles. The performance of the reagent may be affected and it may only work for the first week or so. It is safer to use a new vial. 

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Lipofectamine® MessengerMAX™ Transfection Reagent

We also observed some toxicity in our initial experiments with mRNA. However, incorporating the proper 5’ UTR and 3’ UTR sequences into the template used for in vitro transcription quickly resolved it. Another key factor was the purity of the mRNA. Ensure that the OD 260/280 ratio is between 1.8 and 2.1. The quality of the mRNA can also be determined by running a small amount on a gel to check for the proper size. In some scenarios, it might be best to also incorporate chemically modified nucleotides in the transcription reaction. Another reason for toxicity may be a result of the cells themselves; if the cells are at too low a density then there will be significant toxicity (ideal viable cell density on the day of transfection is between 70 and 90% confluence).

We have seen this as well throughout our labs. Since we identified this issue, we have implemented a standard best practice of only utilizing cells for transfection between 5–20 passages, because at a low passage there is very low transfection efficiency and at a higher passage the optimal dose of transfection shifts. We have also implemented the standard use of a positive control within each transfection experiment to quantitatively determine and track efficiency from week to week.

There are reasons that can influence expression after transfection, but before troubleshooting all the possibilities, a transfection experiment with a positive control reporter and the Lipofectamine® MessengerMAX™ mRNA Transfection Reagent could be the solution. If this does not yield good results, it might be best to try an alternative delivery solution or different cells. However, if this gives acceptable results, the next step would be to try the mRNA of interest with the MessengerMAX™ reagent. If there are expression level concerns at this point, it might be the mRNA that is being used, and troubleshooting from this perspective might be needed. For example, some questions to ask would be: Is there a 5’ cap? Is there a poly(A) tail? Is the mRNA pure? Do I get a single band on a gel? Was the DNA template clean? 

Lipofectamine® RNAiMAX Transfection Reagent

Freezing can alter the integrity and composition of the lipid particles. The performance of the reagent may be affected and it may only work for the first week or so. It is safer to use a new vial. 

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Check to see if antibiotics were added to the medium during transfection, as this can cause cell death. Test serum-free media for compatibility with Lipofectamine® RNAiMAX Reagent.

Invivofectamine® 2.0 Transfection Reagent

This is not unusual; just vortex gently to resuspend the pellet, and use as recommended.

Check for column overload. We do not recommend using more than 15 to 30 mL per column. We recommend splitting the sample over 2 columns and re-spinning.

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