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Q1. Are cell density (% confluence) and passage number important considerations for transfection?

A: Yes, cell density is an important parameter in influencing transfection efficiency. If the seeding density is too low, some cytotoxicity may be observed. If the cell density is high, lower than expected transfection efficiency may be observed. Both issues may be easily resolved by either decreasing or increasing the quantity of complexes added to the culture. LipoGeneTM provide excellent transfection efficiencies at confluence between 70 and 90%. Some toxicity may be observed at lower confluence but may be alleviated by decreasing quantity of complexes or removing the complexes after 4-6 hours incubation and refreshing the media.

Passage number may affect transfection experiments. Excessive numbers of passages may decrease transfection performance. We do not recommend splitting cells for more than 20-30 passages. 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.


Q2. Can antibiotics be used in media during transfection?

A: Yes, antibiotics (penicillin-streptomycin) can be used in the medium during transfection. We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For certain cell types that are sensitive to transfection or have toxicity issues, omitting antibiotics may improve results, however. For stable transfections, wait at least 48 hours after transfection before adding selective antibiotics.


Q3. Why would the expression level of my gene in transiently transfected cells be greater than those that are stably transfected?

A: Expression in transiently transfected clones is typically higher because transiently transfected cells can have up to hundreds of copies of the plasmid containing the gene of interest. Stably transfected clones usually harbor 1-2 copies integrated into the genome, and hence have lower levels of expression. Sometimes, the lower expression level in stably transfected cells is due to adverse effects of the recombinant protein on the cell when expressed constitutively.


Q4. Can I use the same amount of any transfection reagent for different cell lines?

A: The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use.


Q5. Can I use LipoGeneTM for transfection of suspension cells, other hard-to-transfect cells, and primary cells?

A: Yes,you can use LipoGeneTM for transfection of suspension cells, hard-to-transfect cells, and primary cells


Q6. What is the appropriate way of adding lipofectamine 3000 to cells?

Is it added after removing media from each well, and then adding the lipo and DNA mixture dropwise to the well, incubating for 6 hours and then supplementing the cells with complete media (10% FBS)

Or is it appropriate to add the lipo and DNA mixture directly in the media(without removing media from the attached cells)

Do both these methods affect transfection effeciency?

A: In my experience, it is appropriate to mix the lipo and DNA firstly, then the mixture should be added drop by drop into the media. Change the media before transfection would be very helpful.


Q7. How can I improve my HEK293 cell transfection?

I use HEK293 cell lines and trying to transfection D3R.

My protocol is 2.5 microgram DNA is added to 100 microliter serum free media, then added 18 microliter frozen polyethylimine (PEI) and vortex it several time. Incubated for 15min. Then added 900 microliter serum free media to it. Then this DNA solution is transfer into 100 cm cell culture dish which contains 9 ml serum media. Then 4hour incubation. After 4 hours change this serum media to 9 ml serum media.

But this process show extremely low numbers of surface receptor. Can any one give me a proper way.


A: I think the PEI:DNA ratio you used is too high, I suggest you to try 2-2.5 ratio. Besides, the cell state and confluence are very important for a high efficiency transfection, you should replace medium before transfection and ensure the cell confluence between 70-80%.


Q8. What are the ways to troubleshoot transfection using lipofectamine 3000 reagent and prevent cells from dying due to transfection?

I am performing transfection on MCF7 cells using lipofectamine 3000 (invitrogen) but unable to get any transfectants, instead the cells are dying after incubation with reagent and DNA.

A: In my experience, the cell state and confluence are very important for high efficiency transfection. So, you should make sure that your MCF7 cells is in a good cell state. CurePlasma from Genemedi is a broad-spectrum anti-mycoplasma reagent, which may help you to effectively cure mycoplasma contamination within two weeks without affecting your cell viability, you could find more information in the following website: https://www.genemedi.net/i/cureplasma-mycoplasma-elimination-reagent. Besides, replacing medium before transfection and ensuring the cell confluence between 70-80% may be required for high transfection efficiency.


Q9What is the speciality of Opti-MEM media? Can I use DMEM with 5% FBS instead of Opti-MEM media? Why do people use Opti-MEM media for siRNA biology?

A: Opti-MEM is very good for transfection, but DMEM with no addition of serum is also OK for siRNA transfection, which is much cheaper and very convenient.


Q10. Does anyone have a good protocol for puromycin selection for pSpCas9(BB)-2A--Puro transfectants?

I have obtained pSpCas9(BB)-2A-Puro vector from Dr. Zhang's lab via Addgene.

This vector should allow me to select the transfectants by puromycin. However, since pScCas9 is a gRNA producing plasmid, I should select not stable transfectants but transient ones. I am wondering if anyone has experience with this vector, and any suggestion for timing and dose of puromycin selection.

A: We always select cells with puromycin at 48h post-transfection. And you’d better perform a puromycin kill curve on your cells first to check what concentration of puromycin kills 100% of untransfected cells. Normally between 1-10ug/ml works.


Q11How can I optimize the transfection efficiency using lipofectamine 3000(invitrogen)?

Cells are dying after transfection due to toxicity of either DNA/ lipofectamine reagent

What specific media should be used while diluting the lipofectamine reagent (recommended optiMEM or any reduced serum medium). And, how does it affect the efficiency?

A: what cell line do you transfect, some cell lines are more sensitive to liposome. If cytotoxicity is observed, you can try these two methods: 1. reduce the liposome usage, 2. increase the cell confluence before transfection.


Q12What is the best fixation for fluorescence microscopy of GFP tagged proteins? I have had some very faint signals from cells transfected with GFP tagged proteins and fixed for ten minutes in 4% paraformaldehyde. Is this fixation too harsh for GFP?

A: We always use 4% paraformaldehyde (in PBS) to fix cells for immunofluorescence at room temperature for 20min or 4 ℃ overnight. 10 minutes in 4% PFA is OK for fixation. I think there are other problems, such as transfection efficiency or the abundance of protein tagged by GFP.


Q13Difference between FBS and FCS? 

Hi, I want to know whether there is any difference between FBS (fetal bovine serum) and FCS (fetal calf serum). Most people say it is the same thing. However, I plan to culture NIH/3T3 cells and ATCC recommends using FCS and not FBS. https://www.atcc.org/products/all/CRL-1658.aspx#culturemethod. Does anyone have experience working with NIH/3T3 or Balb/3t3 cell line and what is the best way to transfect them?

A: There is no difference between FBS and FCS, it is the same serum with name preference. However, the 'F' for fetal is very important, and catalog numbers from different companies are critical for cell culture.


Q14Hi everyone! What is the difference between MEM and Opti-MEM media? Which one is more effective for plasmid transfection into eukaryotic cells?

A: Opti-MEM may be the best option for transfection, but DMEM without FBS also works very well, usually no significantly difference can be oberved.


Q15Is there an alternate for Opti-MEM for lipofectamine transfection? Can I use MEM with low supplemented FBS?

A: Opti-MEM may be the best option for transfection, but DMEM without FBS also works very well, usually no significantly difference can be oberved.


Q16How many hours of incubation time after transfection is best to harvest cell culture?

A: The best harvest time depends on the experiment design, nucleci acid type, cell type and expression efficiency, many factors need to be taken into account. I assumed you want to ask the highest expression time point. Usually, you can harvest cell sample at 36-48h if you transfect siRNA, 48-72h if you transfect plasmid for gene expression.


Q17Breast cancer cell line MDA-MB-231 transfection

Recently we tried to transfect a plasmid into MDA-MB-231 and MCF-7 cell lines. After transfection we found that its transfection efficiency is different from other cell lines. MDA-MB-231 cell line is difficult to transfect with lipofectamin2000, after optimized transfection condition we only reached about 1% transfection efficiency. Has anyone performed transfection to this cell line with lipofectamin2000? Can anyone offer suggestions?

A: MDA-MB-231 is truely hard to transfect, one choice is to use lentivirus or adenovirus, their infection efficiency is much better.


Q18Why I can't see any DNA band on Agarose gel?

I have transformed my cloned plasmid DNA into bacteria and cultured bacteria. Then, I extracted DNA from bacteria and run in 0.5% agarose gel. But I am not seeing any DNA band except ladder.

(In nanodrop, the conc. was 86ng/microliter, but 260/280 ratio was 1.62)

To check the gel, I had run another DNA besides this DNA. But I found the band of another DNA. What could be the problem?

A: I think your plasmid may lose during plasmid amplification in bacteria, in this case, slowing down the rotation speed would be helpful. Or the plasmid was not extracted from bacteria, you should optimize your plasmid extraction method.


Q19Has anyone tried siRNA transfection with Lipofectamine-3000? Protocols/suggestions are appreciated.

A: It’s very common to transfect siRNA with lipofectamine-3000. You can just follow their instruction manual.

a. Propagate your cells as usual. The day before transfection, plate the cells in a 10cm dish such that the cells reach 70-80% confluency the next day.

b. The day transfection (Per DNA-Lipo P3000/Lipo 3000 Reagent complex):

siRNA + 6ul of Lipofectamine P3000; 7ul of Lipofectamine Reagent;

Genemedi launched a product of Lipogene, comparable to lipofectamine 3000, but with fewer cytotoxic effect. 


Q20What are the advantages or disadvantages of using the CRISPR/Cas9 vs. RNAi?

A: CRISPR is labor intensive, mediating gene knockout by destroying the genome, needing to screen the homozygous cells, and can't avoiding potential off-target effects, while RNAi contains siRNA and shRNA, mediating gene knockdown by promoting the degradation of mRNA, but the knockdown efficiency is unpredictable, and you need try several siRNAs or shRNAs to get the sequences with high efficiency. If you want to create a stable cell line, siRNA is a better choice for you.

Genemedi launched a product of Lipogene, suitable for siRNA transfection, comparable to lipofectamine 3000, but with fewer cytotoxic effect. 


Q21How can I make a stable HEK293 cell line using pcDNA3.1?

A: To make stable HEK293 cells, we always transfect HEK293 cells with the available transfection agent (we generally use Lipogene, comparable to lipofectamine 3000, but with fewer cytotoxic effect. You could find more information on this website: www.genemedi.net/i/lipogene-transfection-reagent).

Take a 6-well plate as an example, the detailed protocols are as follows.

1) Seed cells one day in advance to make sure the confluency can be 70%-80% before transfection.

2) Transfect the required amount of DNA using the LipoGene as the manufacturers protocol suggested.

3) Change the media 6 hours post-transfection to achieve higher transfection efficiency.

4) 48 hours post-transfection, select cells with antibiotic in your vector backbone. The overexpression efficiency can be determined with Q-PCR or Western blot.

5) Then you should perform you experiments as soon as possible, for this method is just for transient cell lines.

If you want to establish stable cell lines, virus mediated transfection is needed, Lentivirus is really an efficient way to generate stable cell lines. Integration into host cell genome, it mediates long-term and stable expression of exogenous genes with a simple system for vector manipulation and production. Genemedi got a rich experience in lentivirus production and titration, you could find more information on https://www.genemedi.net/i/lentivirus-packaging.


Q22Improving a low PEI HEK-293 transfection efficiency?

A: I think the low transfection efficiency is because you added too little PEI, your added 194uL 400ug/ml DNA should corresponding to 155-194ul 1mg/ml PEI, the ratio should be 1:2~1:2.5. Besides, 50% confluent may be the reason for observed cytotoxicity, I suggest you to transfect the 293 cells with 70%-80% confluent.


Q23.Can anybody give me exact protocol for siRNA transfection using RNAiMAX ? what should be the ratio between siRNA oligos and RNAiMAX ?

A: We always use 60ul RNAiMAx to transfect about 20nM siRNAin 10cm dish. However, siRNA transfection efficiency may vary from the cell line you're using. I suggest you carry out a preliminary experiment to test the best ratio of RNAiMAX and siRNA, then use the optimal concentration to transfect.


Q24Has anyone used Lipofectamine 3000 for plasmid transfection in HepG2 cells?

A: Compared to Lipo 2000, Lipo 3000 is more suitable for the delivery of plasmid DNA into Hep G2 cells, with much higher transfection efficiency. And reverse transfection is indeed easy to induce cytotoxicity than traditional forward transfection. To reduce the cytotoxity and increase the transfection efficiency, LipoGene is a good choice, for with its higher efficiency (>80% with 293 cells) and fewer cytotoxic effects.


Q25How do you co transfect two plasmids?

A: We always mix the two plasmids together with the same molar ratio to transfect and they worked very well.