How metaphor agarose can be used to genotype Indels introduced by single gRNA?

Dear all, We have introduced double stranded break in our gene (exonic) using a single gRNA. We have also checked efficiency of the cut by T7E1 assay. Now we are trying to genotype invidual clones...

05 June 2018 5,318 3 View

How can I tag mRNA by cloning inside a cell?

Hi I am interested to check a particular mRNA and a protein interaction/ localization inside cells. For this reason, I want to tag the transcript. Beside RNA-FISH or any such hybridization...

10 November 2017 8,697 0 View

Any suitable protocol for Ni-NTA based pull down and interaction study?

Hi..I have over-expressed a protein (tagged with His) via retroviral transduction method in THP-1 cells. Now I am interested to check if there is any binding partner with my tagged protein in a...

11 December 2016 2,637 4 View

How can I separate ribosome-free cytosol using sucrose cushion?

I intend to separate ribosomal and non-ribosomal fractions of cytosol using 20% sucrose cushion. I have already run the cell lysate on top of the 20% sucrose solution for 2 hrs at 1,39,000 g. I...

05 June 2016 1,102 2 View

How can I search the expression of particular set of genes in NCBI GEO database?

I am interested in searching the gene expression profile of a particular set of genes in a specific disease condition like atherosclerosis from NCBI GEO database. As I have almost no background in...

06 July 2015 615 7 View

Why CD11b and CD14 mRNA expression is getting down after 6hrs of rest?

I have converted THP1 monocytes to macrophage using 5 ng/ml PMA for 48 hrs in complete RPMI1640.PMA Containing media was discarded and cells were washed with sterile DPBS for 3 times and given...

03 April 2015 5,086 2 View

Why are my PMA differentiated THP1 cells not responding to LPS induction?

I have differentiated THP1 monocytes into macrophage using 5 ng/ml PMA for 48 hrs. Then after washing twice with DPBS i have kept the diffrerentiated cells for 3 hrs in serum free media. Then I...

02 March 2015 1,298 8 View

Why are my THP1 cells forming clumps?

I am culturing THP1 cells in RPMI 1640 (as advised by ATCC formulation, except 2ME). Unfortunately my cells are forming clumps a few hours after seeding (at 1* 10^5 cells/ml) before even going to...

02 March 2015 154 10 View

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02 March 2021 3,060 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 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

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 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

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 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

What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View