How to mutate a DNA sequence to stop binding to its specific primer?

I am doing reverse transcription using a semi-suppressive PCR therefore I introduce a template switching oligo at the 5' end of my transcript which is complementary to the PCR handle at the 3'...

09 October 2018 7,558 2 View

What are the most suitable reference genes for human GOI qPCR?

I want to carry out a qPCR for a human gene of interest and I need to decide on a suitable reference gene. For the past few years I have been using Actg1 for my murine studies and therefore I...

06 July 2014 1,656 7 View

Any common protocol for inflammatory stimulation of murine endothelial cells in vitro?

11 December 2013 4,729 9 View

How do people derive BMDMs from bone marrow cultures?

Do you culture for 6 or 7 days, do you plate a certain number of cells on Petri dishes, do you wash the media on day 3?

10 November 2013 4,397 12 View

Can the measurement of cytokine levels according to their half lives be useful?

A question of cytokine kinetics. In an activation assay, we usually want to measure effects of our treatment (whatever this might be) on pro- or anti-inflammatory cytokines. Most commonly...

10 November 2013 7,766 4 View

MPO activity assay - can anyone help?

I homogenized murine lungs in 0.5% w/v HDAB in 50mM sodium phosphate buffer to perform a MPO activity assay. The protocol I do have instructs that I should use 20ul of my samples with 160ul TMB...

09 October 2013 2,105 4 View

How fast should an ELISA plate be read?

I finished an ELISA assay and read it to discover two hours later that I saved the 630nm data which I used to correct my 450nm data. I immediately measured the plate again but I'm not sure whether...

08 September 2013 1,312 20 View

qPCR data representation

I have some qPCR data which I want to represent in graphs. I used to use the DCt values but the problem is that they are reciprocal to the expression changes (high DCt values means low expression)...

06 July 2013 393 2 View

GFP DNA sequence

I want to design qPCR primers for eGFP so I've looked up the literature for its DNA sequence but the outcome was rather confusing; too many "GFP" sequences that I now don't know which one is the...

06 July 2013 4,585 2 View

Which transcription factors are activated early in response to inflammatory stimuli?

05 June 2013 8,018 35 View

Why does blocking with my immunogenic peptide cause nuclear fluorescence in ICC?

Hi all, I am validating a polyclonal antibody raised against human ANT4, through WB and ICC. In most of my controls, the antibody shows very little off-target binding, but when I pre-incubate the...

03 March 2021 5,999 3 View

Which of the Covid-19 Vaccines would elicit the best immunoprotection and why?

I want to know which of the available Covid-19 Vaccines would elicit the best immunological response and why

02 March 2021 1,388 3 View

Does Napabucasin soluble in dichloromethane and in ethanol?

I need to prepare a solution of Napabucasin (inhibitor of cancer cell stemness) in dichloromethane. The drug is expensive, so maybe somebody already tried to do it. If it is soluble, what is the...

02 March 2021 9,945 3 View

Help request in CST microwave?

im working now on TPV solar cells and metamaterials and i realy need our help in CST microwave i have a tpv cell simulation and i want to extract particullarly the absorbtion of only the active...

02 March 2021 2,246 2 View

Dapi fluorescence protocol for colon cell lines ?

28 February 2021 2,534 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

Has anyone had luck using cell lines other than HEK 293 (such as COS1) to package lentivirus?

I am looking to package lentivirus in endothelial cells but I am opposed to using 293 cells due to personal ethics regarding the source. I have done some reading and saw that COS1, COS7, & CHO...

25 February 2021 3,030 1 View

Unknown debris sheets observed in cell culture flasks?

Hi, We culture neural progenitor cells in T175 culture flasks coated with PLO-Laminin. We have successfully grown these cells with our protocol for months. Recently, we observed large sheets...

25 February 2021 3,443 4 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