For CRISPR, transfection or transduction?

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Can FDA-approved drugs be used in other disease setting?

Hi Everyone, I'm exploring the anti-tumor effect of some drugs. I'd like to test some FDA-approved drugs which are not used to treat cancer. My question is, if there is any anti-tumor effect for...

14 December 2020 1,464 1 View

How to know if my protein forms a dimer or not?

Hi everyone, The protein I studied is a membrane protein. Some study indicates that there is possibility that this protein can form a dimer. I did standard western blot for this protein and my...

20 October 2020 6,935 7 View

How to ensure a marker antibody won't activate T cells and affect the result?

I'm recently buying some anti-CD3 and anti-CD28 to activate T cells. I also notice some of this clones have been conjugated with fluorescence for the purpose of labeling T cells for flow...

06 October 2019 7,129 3 View

Why my gene in single-cell sequencing showed zero value?

I have many single-cell sequencing data. I checked my gene of interest in these data. However, all of these single-cell sequencing data showed that my gene has no expression, no matter in immune...

03 January 2019 1,707 3 View

Why can't I detect the surface protein by western blot?

I was doing an over-expression experiment of a gene for a year. It is a surface protein. The biggest trouble is that I can never detect the gene overexpression by western blot. Interestingly, when...

29 November 2018 6,311 3 View

Can I use NEB mutagenesis- substitution method to clone sgRNA into the vector?

Can I use NEB mutagenesis -- substitution method to clone sgRNA into the vector? I felt it's much easier. Just design the primers containing the sgRNA bp, conduct PCR and do a ligation. And most...

08 March 2018 4,991 1 View

What's the best way to eliminate Mycoplasma?

Recently check the cell lines, 60% of the cell lines are infected with Mycoplasma. Some common antibiotics for treating Mycoplasma were used to treat cells for 3 weeks, but mycoplasma is still...

14 February 2018 9,511 2 View

Why using RecA- bacteria strain?

The growth instruction for a CRISPR plasmid said that the plasmid need RecA negative E.coli strain like Stbl3 to grow. What is special for RecA negative strain? Why the clone need RecA negative??

19 January 2018 5,047 6 View

How is heavy chain and light chain of an antibody connected?

I have CHO cells that transduced with two vectors expressing light chain and heavy chain of an antibody respectively. How do they automatically associate with each other and form antibody? What...

27 December 2017 5,668 4 View

To stimulate T cells, secreted antibody is better or bound antibody is better? why?

Among anti-CD3, CD28, OX40, 41BB, which is better to be bound antibody and which is better to be secreted antibody in order to stimulate T cells? And why there is such a difference of function...

14 December 2017 3,323 1 View

Can linear DNA be used as template for site-directed mutagenesis?

Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...

03 March 2021 401 4 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

PUC19 as a transfer plasmid for lentiviral delivery?

Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...

28 February 2021 4,868 2 View

Does RNAiMAX transfection reagent work in serum free medium?

I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium. Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in...

26 February 2021 10,041 3 View

How do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?

I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...

25 February 2021 6,635 5 View

What is a good reagent for lentiviral plasmid transfection in HeLa cells?

I have been using Lipofectamine 2000 but was hoping there may be a better protocol or one for something like Lipofectamine PLUS reagent.

24 February 2021 1,633 4 View

Why the cells die after stable transfection?

I tried stable transfection in the U87 cell line with the sgRNA-Cas9 All-in-One lentivector. Cells were cultured in a 6-well plate, 4ug plasmid was transfected after 24hs when the cell confluence...

22 February 2021 2,864 4 View

Is there a method for evaluating nanoparticle adhesion to epithelial tissue in vitro?

I want to measure how well nanoparticles can transfect cells based on NP adhesion properties. In the literature I find people do it in animal studies, and epithelial adhesion helps in transfection...

21 February 2021 7,791 3 View

Petr Muller

Hi I'm using lentiviral CRISPR V2 vector from Zhang's lab. https://www.addgene.org/52961/ I'm getting much better results with transduction than standart transfection (Lipofectamine). The puromycin selection after transduction results in almost 100% succes in gene knockout (checked by sequencing). I believe that lentiviral expression, unlike transfection, can provide a more even and more appropriate expression of large proteins, such as CAS9. However, puromycin selection is required to remove uninfected cells.

Michael Davis

It really depends on your cells and methods of transfection. If you have an easily transfected cell line, there is no need to go through the effort of making virus for transduction. Test you transfection efficiency with a GFP expressing vector. See how many of your cells are green. If it is a high percentage, you are good to go. Good luck.

Ibrahim T Hagag

Transduction!