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

In there any report on Lipofectamine RNAiMAX mediated transfection of CRISPR-Cas9 plasmid in THP-1 cells?

02 March 2017 4,713 1 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

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

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

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 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

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

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 3 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

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View

Can anyone provide the Support vector regression code structure of LIBSVM MATLAB or python ?

Please, if Gaussian Parameter optimization is possible in this code structure

28 February 2021 278 3 View