Do mouse primary hepatocytes survive in transfection solution without serum ?

hello, i am planning to transfect mouse primary hepatocytes with miRNA in order to isolate microsomes to study the activity of my enzyme of interest. Therefore, I wondered if the primary...

07 August 2019 2,067 2 View

How to calculate amount of beta-glucuronidase in powder from 0.1 M sucrose?

Hello, how can I calculate the actual amount (in mg) of beta-glucuronidase from supplied white powder lyophilized from 0.1 M sucrose plus 1 mg sucrose per mg protein ? On the delivered vial it is...

09 October 2018 4,766 0 View

How to measure endogenous miRNA expression from 2 different body compartments ?

Hello RG community, My task is to measure the expression of several microRNAs from total RNA isolated from human body sera. I have a number of TIPS patient (suffering from liver cirrhosis) and...

07 August 2018 5,142 1 View

What is best reference gene when quatifying circulating miRNA from TIPS patient serum ?

Hi there, I received extracted whole RNA from TIPS patient serum and negative healthy controls. All samples contained spiked-in SV40 miRNA already as a reference gene. I performed miRNA reverse...

03 April 2018 3,960 5 View

Who could provide primers for quantification of retinoid X receptor alpha (RXRA) mRNA?

02 March 2018 1,051 5 View

Is glucuronide CDCA-24G soluble in water or in any other solvent?

Hi guys, I have a question about glucuronide CDCA-24G, maybe someone has already worked with it. I want to perform an experiment in hepatocytes which I have extracted from mouse livers. I do not...

01 February 2018 1,856 2 View

How can I achieve to mutagenize a restriction site in a 6.5 kb plasmid?

Hi all, I tried to insert a restriction site in a 6.5 kb plasmid having only six endogenous restriction sites in the MCS. I already tried several primer setups but all failed. What could I...

10 November 2017 3,495 10 View

Is the number of clone picking to find integrated restriction site in plasmid equal to general process of insert screening ?

08 September 2017 8,176 4 View

How to exclude interactions of miRNA with the Luciferase reporter plasmid?

Hi all, does someone have experience with studying the interaction of microRNA (miRNA) and its target sequence cloned into a Luciferase Reporter vector? Actually using pGl3-Basic vector from...

07 August 2017 3,539 5 View

How can I use the Neubauer cell counting chamber?

08 September 2016 8,917 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

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

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

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

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 3 View