May I use anti-GFP antibody as an isotype control for ChIP?

I plan to perform ChIP experiments with wild-type mouse cells (no GFP transgene) using an anti-protein antibody raised in goat. May I use an anti-GFP antibody of the same isotype raised in the...

01 February 2020 2,469 1 View

Do B lymphocytes respond to foreign DNA during transfections?

During transfection of B cells with plasmids/linear DNA fragments, do the large amounts of foreign (bacterial) DNA sequences activate signalling cascades, like that of TLR9 for CpG-rich sequences...

07 August 2018 2,230 1 View

Why is Lamin B1 not showing up in primary B cells' western blot?

06 July 2017 6,957 0 View

Peak shifts in cell proliferation assay using CFSE?

I am doing a time course experiment to study cell divisions. I have carried out the experiments in dark, given the light sensitivity of CFSE. I have analysed stained and unstained samples at every...

31 December 2016 7,763 11 View

What do the numbers after NP (nitrophenyl) mean eg., NP3, NP26, NP33, in the context of antibody affinities?

06 July 2016 6,382 1 View

How to perform nascent RNA quantitation?

Is it necessary to isolate nuclei from cells and use this RNA for qPCR? Or can specific primers be designed in intronic regions so that only unprocessed RNA will be picked, and use whole cell RNA...

05 June 2016 1,991 4 View

Why would a double-cut plasmid with ligase give colonies?

I am using a plasmid vector of about 8kb for cloning. After cutting with EcoRI and BamHI, I performed a ligation with T4 DNA ligase and transformed in DH5-alpha, and found that there were as many...

10 November 2015 8,216 11 View

Can the gene of interest be cloned after a viral 2A peptide?

I am interested in making a bicistronic system using viral 2A peptide to clone my protein of interest. The usual order of cloning is GOI-P2A-GFP (for example). Since the cleavage site is at the C...

07 August 2015 8,679 2 View

Why do I get a smear on the gel after the re-amplification of a PCR amplicon?

I have observed multiple times that if I use a PCR amplicon (with / without PCR cleanup) as a template for a subsequent PCR with the same primers, I get a smear on agarose gel. The size of the...

04 May 2015 9,447 20 View

How do I resuspend Trizol protein pellets?

I have been resuspending protein isolated by Trizol reagent in 1% SDS and storing at -20 degrees C. But everytime I need to use the protein for quantification, electrophoresis, etc., I need to...

04 May 2015 6,778 2 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

Which is a lucid standard comprehensive book to guide on psychological scale construction and its standardization?

02 March 2021 7,082 3 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

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

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