How do I culture more CD11c+ BMDC from mice?

Hello, I am new to harvesting BMDC and my most recent experiment had 45% CD11c+ cells that were viable (also many dead cells, only 20-30% viable). I followed the method from Madaan A, et al....

06 July 2018 319 3 View

How many times can you strip a western blot?

Hello, How reliable are western blots after they have been stripped? Does the buffer strip all lanes equally? If you load 20 ug onto a gel and strip twice, how much would be left? I'm trying to...

01 February 2018 4,840 3 View

How much EDTA should be used for flow cytometry purposes?

Hello, I've been perusing the literature and a variety of protocols use EDTA concentrations from 0.03-5 mM EDTA in their buffers for isolating immune cells from the lung and/or BALF for flow...

31 December 2017 2,134 3 View

Why does the type of collagenase matter for lung digestion?

Hello! I am attempting to analyze cell populations in mouse lung homogenate using flow cytometry. I have seen a variety of protocols that use a range of collagenase and DNAse concentrations,...

09 October 2017 4,684 4 View

When propagating laboratory strains of Influenza, is ultracentrifugation required for purification?

07 August 2016 8,045 0 View

How do you use cell lysates for studying enzyme kinetics?

Hello, I'm trying to determined the Vmax, Km, etc...for a membrane bound short chain dehydrogenase. I am planning on using cell lysates as a source of the enzyme. Do you know of any protocol to...

03 April 2016 1,925 5 View

What is the best way to determine the effect size?

How can you use preliminary data and/or previous research to determine your effect size so that you can estimate power and sample size required for future animal studies/clinical studies?

11 December 2015 3,300 3 View

What is a solid method to separate nuclear and cytoplasmic proteins from lung tissue?

I've been using commercial kits to separate cytoplasmic and nuclear proteins. I noticed that many advise against using their kits for frozen tissue because the nuclear membrane can break and leak...

06 July 2015 9,735 1 View

Can anyone help with disappearing plasmids?

I want to know if anyone has ran into this problem before: I'm making a deletion construct and I have positive results in colony screening. When I miniprep the plasmid after overnight culture, the...

07 August 2014 4,079 8 View

What is your perception on using visual analysis or quantitative approaches to evaluate data from single-case research designs (n of 1 trials)?

Results of single-case research designs (i.e., n-of-1 trials) are often evaluated by visually inspecting the time-series graph and computing quantitative indices. A question our research team is...

03 March 2021 687 1 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

PACYC PCR not working after several attempts. Would anyone have any suggestions?

So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however...

02 March 2021 1,146 2 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

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 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

Runs of nucleotides in Sanger sequencing?

I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...

27 February 2021 547 3 View