Transfection Reagent FAQs

Questions

  1. What recommendations do you have for selecting a transfection reagent?
  2. What is the stability of transfection reagents?
  3. Is cell density (% confluence) and passage number important considerations?
  4. Can I use the same amount of any transfection reagent for different cell lines?
  5. I am working with well sizes different than your protocol. How do I scale up or scale down my transfection reaction?
  6. Where can I find cell line-specific transfection protocols?
  7. Is there a place I can find references from other researchers who have used your reagents?
  8. Can antibiotics be used in media during transfection?
  9. I am creating stable cell lines. What recommendations do you have for working ranges?
  10. What types of molecules can be transfected using Invitrogen transfection reagents?
  11. Can transfection reagents be used to co-transfect plasmids? Can I co-transfect plasmids and siRNA?
  12. Which transfection reagent does Invitrogen recommend for RNAi applications?
  13. Why would cellular expression levels of my gene in transient transfectants be greater than observed in stable, antibiotic resistant transfectants?
  14. What are your recommendations with regards to endotoxin-free DNA for transfections?
  15. How do you estimate the amount of DNA using A 260 readings? What does A260/A280 mean?
  16. Sometimes I see a small granular-like precipitate on my cells (microscopically) following addition of the transfection reagent:DNA complexes on my cells. Why does this happen and will I see a decrease in transfection performance?
  17. Is Lipofectamine™ 2000 the same as Lipofectamine? What about Lipofectamine™ PLUS and Lipofectin®?
  18. Why do I see low transfection efficiency?
  19. Why do I see cytotoxicity after performing transfection?
  20. Why are my transfections not reproducible?

Answers

  1. What recommendations do you have for selecting a transfection reagent?

  2. What is the stability of transfection reagents?
    Invitrogen’s transfection reagents are shipped on wet ice and should be stored at 4 °C. Invitrogen guarantees the performance of the product, if stored and handled properly, for six months following purchase. We do not recommend freezing transfection reagents as this usually decreases transfection performance. All tubes are labeled with an expiration date that is two years after date of manufacture.

  3. Is cell density (% confluence) and passage number important considerations?
    Yes. Cell density will affect transfection performance. For example, Lipofectamine™ 2000 provides excellent transfection performance at a confluence of 90% or greater, while slight toxicity may be observed at confluencies lower than this. We recommend using Optifect™ for cell densities <70% confluence.

    Passage number may affect transfection experiments. We recommend splitting and plating cells consistently. Excessive numbers of passages may decrease transfection performance. If transfection performance declines and cells have been in culture for a long time or excessively/improperly passaged, we recommend that you restart your cultures with a new vial of cells from liquid nitrogen.

  4. Can I use the same amount of any transfection reagent for different cell lines?
    First, each of our transfection reagents is provided with an optimized protocol, and the amounts of reagent needed per well may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use.

    Secondly, in most cases, the protocol that is provided with the product will provide you will an optimal range of transfection reagent to use per well. During product development, this range was determined to work well across a variety of cell lines. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary. Please contact Technical Support for further assistance with optimizing your protocol.

  5. I am working with well sizes different than your protocol. How do I scale up or scale down my transfection reaction?
    Each of our transfection reagent protocols provides a table for scaling up or down transfections. An example from the Lipofectamine™ 2000 protocol is posted below.

  6. Where can I find cell line-specific transfection protocols?
    Our Cell Lines Database includes protocols and citations for hundreds of cell lines and is located on the Technical Resources page. Featured protocols for RNAi transfections can be found here.

  7. Is there a place I can find references from other researchers who have used your reagents?
    Our Cell Lines Database is updated to include papers from researchers who have published their results and referenced Invitrogen transfection reagents. This information can be found via the online catalog page (click on the blue book icon under “Tech. Docs.”) or via the Cell Line Database. R&D also recommends www.highwire.org as a search engine to find a large selection of up-to-date research articles using Invitrogen transfection products. Simply include “Lipofectamine 2000” (or other Invitrogen product) and your cell line/application of interest in your search criteria.

  8. Can antibiotics be used in media during transfection?
    We discourage using antibiotics during transfection. Higher cell death can occur when antibiotics are present during transfection. Even though penicillin and streptomycin are not toxic to eukaryotic cells, during transfection the cell permeability increases so that much higher levels of antibiotics get into cells. For stable transfections, wait at least 72 h after transfection before adding selective antibiotics.

  9. I am creating stable cell lines. What recommendations do you have for working ranges?
    Geneticin® Selective Reagent, is the Invitrogen brand name for G418. It is the most popular selection marker for mammalian cell culture and typically used at a concentration of 600 mg/mL. Blasticidin S HCl, Zeocin™ Selective Reagent and Hygromycin can also be used and each have distinct advantages (see the table below).

    Read more information on the Geneticin® Advantage.

    Recommend Eukaryotic Reagents

  10. What types of molecules can be transfected using Invitrogen transfection reagents?
    Invitrogen’s cationic lipid transfection reagents can be used to transfect DNA, siRNA, Stealth™ RNAi, dicer-generated siRNA pools or plasmids containing shRNA cassettes. Oligonucleotides, proteins, and RNA can also be transfected. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb.

  11. Can transfection reagents be used to co-transfect plasmids? Can I co-transfect plasmids and siRNA?
    It is possible to co-transfect plasmids and co-transfect plasmids and siRNA using Lipofectamine™ 2000. Please click here for protocol information.

  12. Which transfection reagent does Invitrogen recommend for RNAi applications?
    For most cell types, Lipofectamine™ 2000 is recommended for transient delivery of Stealth™ RNAi, siRNA, dicer-generated siRNA pools and plasmids containing shRNA cassettes (Figure below). We recommend using Oligofectamine™ when transfecting siRNA into HeLa cells.

    Rapid Procedure Lipofectamine 2000

  13.  Why would cellular expression levels of my gene in transient transfectants be greater than observed in stable, antibiotic resistant transfectants?
    Transiently transfected cells have a higher copy number of the gene (hundreds per cell) and therefore higher levels of expression result. The level of expression in stably transfected cells depends on the number of integrated copies, which are usually in the order of 1-2 per cell.

  14. What are your recommendations with regards to endotoxin-free DNA for transfections?
    Some researchers prefer endotoxin-free DNA for their transfection experiments if they are working with cells that are particularly sensitive to endotoxins. To prepare endotoxin-free DNA, Invitrogen provides a number of nucleic acid purification kits called “PureLink™ HiPure Purification Kits” in Mini, Midi, Maxi, Mega and Giga sizes. For more information, please click here.

  15. How do you estimate the amount of DNA using A 260 readings? What does A260/A280 mean?
    1 A260 unit (plasmid DNA in H2O) = 50 ug/mL

    The extinction coefficient will change if the plasmid DNA is diluted in a buffer other than H2O. This will change the value indicated above.

    Sample calculation:

    Volume of plasmid DNA sample = 100 uL

    Dilution (1/20) = 25 uL of the sample in 475 uL H2O

    A260 of diluted sample = 0.65

    Note: For optimal results, make sure OD values are within 0.1 and 1.0.

    Concentration of plasmid DNA sample = 0.65 x 50 ug/mL x 20 (dilution factor) = 650 ug/mL

    Amount of plasmid DNA in sample = 650 ug/mL x 0.1mL (sample volume) = 65 ug

    An A260/A280 value that is greater than or equal to 1.8 means that the plasmid DNA is pure. A260/A280 readings that are less than 1.8 indicate that the sample may be contaminated with aromatic products (ie. phenol) or protein. Readouts greater than 2.0 suggest that the sample is contaminated with RNA.

  16. Sometimes I see a small granular-like precipitate on my cells (microscopically) following addition of the transfection reagent:DNA complexes on my cells. Why does this happen and will I see a decrease in transfection performance?
    A common reason for seeing precipitate on your cells following transfection is if there is excess EDTA present. We recommend diluting DNA in water or, if TE is preferred, use EDTA concentrations of <0.3 mM. The presence or absence of this precipitate is not indicative of the transfection performance.

  17. Is Lipofectamine™ 2000 the same as Lipofectamine? What about Lipofectamine™ PLUS and Lipofectin®?
    No. These are all different cationic-lipid formulations. Lipofectamine™ 2000 provides the best transfection performance for both plasmid DNA and RNAi delivery over the broadest range of cell types.  Lipofectamine™ is an earlier broad range reagent that was sold by Invitrogen. Lipofectamine™ PLUS is a discontinued transfection reagent, although the PLUS™ Reagent is available and sold separately (Cat. no. 11514-015). Lipofectin® was originally launched in the late 1980s and is considered our very first transfection reagent (see Table below). We continue to sell this product for customers who prefer the older formulation, but recommend that all new customers try Lipofectamine™ 2000 first for optimal performance.

    15 yrs transfection development

  18. Why do I see low transfection efficiency?
    For more information on low transfection efficiency troubleshooting

  19. Why do I see cytotoxicity after performing transfection?
    For more information on cytotoxicity following transfection

  20. Why are my transfections not reproducible?
    For more information on reproducible transfections