How to efficiently transitory transfect ASC52telo cell line?

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How to avoid ulcers in s.c. injected tumors ?

Recently, we have injected s.c. B16F10 tumor cells in BL/6 mice. At around 12-15th day some animals showed ulcerations in tumors , with a big hole in the middle (it looks like a volcano). We have...

01 March 2021 9,938 3 View

What is the standard protocol in doing replicates for C. elegans lifespan assay on solid media (thru worm picking)?

I would like to know what is the standard protocol in the number of worms per plate and number of plates per concentration of extract X in a lifespan assay done in solid media and not automated...

23 February 2021 9,933 1 View

SPSS AMOS: Running my structural model decreases the CFI significantly - trying to account for it results in an unidentified model. What can I do?

Hi, after a thorough EFA and CFA, I've created my structural model (with imputed factor scores) in AMOS and ran it. While it provided me with all path coefficients, I found that the CFI had...

06 January 2021 5,087 4 View

Does anyone know how to fix the "A sample of parameter values was inadmissible"-error in SPSS AMOS when imputing factors?

Hi all, I'm trying to impute factors after testing my model for CMV and therefore included a common latent factor. Unfortunately, I always get the same error: "A sample of parameter values was...

02 January 2021 6,647 5 View

How can I measure the bacterial growth in a wet fabric in a petri dish?

I have to measure the growth of the bacteria in a piece of fabric for my senior design project. I don't know how to measure it. The only way that I know is by the naked eye or by the microscope....

23 November 2020 2,897 6 View

Calculating effect sizes without knowing the sample size by subgroups?

Hello! I am calculating effect sizes for a research synthesis of the literature (e.g. Hedges' g), but many of the original publications do not provide the sample size (N) by subgroups. I see...

13 September 2020 7,587 3 View

Does an oral & Maxillofacial Medicine Specialist or physician have experience prescribing buspirone to a patient with SSRIs-related bruxism?

Does an oral & Maxillofacial Medicine specialist or physician have experience prescribing buspirone to a patient with SSRIs-related bruxism?

09 September 2020 5,952 4 View

Who can help me justifying intervals in my analysis?

Dear researchers, In my study, I have qualitatively (with a bit of quantitative) found results when comparing how cities and states attract international organizations. These results I coin low,...

18 August 2020 9,608 3 View

Will making a hydride acceptor more electrophilic speed up hydride transfer?

I have been searching the literature about this and I have not found an answer. If the atom that accepts a hydride is made more electrophilic, can this speed up the rate of hydride transfer?

29 July 2020 9,944 1 View

Can the energy displayed in Gaussian Calculation Summary be used to compare the stability between molecules?

I want to compare the stability of molecules that have the same number of atoms. I optimized these structures and calculated their vibrational frequencies. Gaussian09 gives a Gaussian Calculation...

18 July 2020 5,243 5 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 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

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 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

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 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

Ahmed E. Dhamad

I used ViaFect (see the link) and worked very well with a cell line that is relatively difficult to be trasfected.

https://worldwide.promega.com/resources/protocols/technical-manuals/101/viafect-transfection-reagent-protocol/

Aadil Javed

Dear Rosa Aledo

The CRISPR plasmids are larger than other plasmids and require stringent conditions for transfection. Lipofectamine has been known to work well with CRISPR plasmids. please read the methods in the following papers to understand the solution. You can focus on lipofectamine.

Article Rapid and Highly Efficient Mammalian Cell Engineering via Ca...

https://www.nature.com/articles/srep05400

https://www.mdpi.com/1422-0067/14/10/19774

Ankur R Dubey

You could use lipofectamine for transfection of plasmids. If you are experiencing problems with efficiency you may try to increase the plamsid and lipofectamine concentrate along with extended incubation time for transfection. This usually gives decent transfected cells.

At beginning you may try with lower cellular density to have better outcomes.