Does anyone have a protocol to transient transfect HEK293 with 2 plasmids?

More Giorgia Croppi's questions See All

Does anyone know if pKD46 system works in E. coli BL21 or DH5alpha to KO a gene?

I need to KO a gene in my bacteria and I'm currently use BL21 strain also for the over expressed one. I've been trying many systems to ko my gene but so far I didn't get any good result and I'm...

03 June 2018 2,089 6 View

Protocol PCR primers narG1950m & narG2050m?

Did someone used these primers in PCR or qPCR? Because I have a problem to perfom the PCR and I obtained the no appropriate band in the samples and NTC. Thanks Giorgia

29 January 2018 1,404 3 View

Non-neurotoxic brain dye for infusion?

Dear all, I'm looking for a non neurotoxic brain dye that I can infuse into the ventricles through osmotic pumps, to be sure that I'm doing the infusion in right way. The mice must survive for at...

29 August 2017 5,509 3 View

Renal sympathetic nerve staining

I know it's commonly done with tyrosine hydroxilase but I was looking for an alternative IHC (less expensive) method. I came to understand silver staining or Luxol staining are possibilities but I...

12 June 2017 6,112 2 View

Which method do I use to find the best gene reference to use un the RT-qPCR?

Hi, I need to find a gene reference to use in the gene expression of cellulolytic genes in Ascomycete. Which gene reference is possible ti use? Or which is the method to find it? Thanks Giorgia

31 May 2017 9,655 1 View

It is possible to express the increase of the expression of a microRNA as the sum of its intracellular content and the one in the supernatant?

I need to quantify the total expression of a miRNA that is contained in a cell and is also released from it in the culture medium. How can I do? Do You know a few articles in the literature that...

12 December 2016 1,073 3 View

RT-qPCR protocol one or two steps?

Is best to prepare the RT-qPCR in one-step or two-step?

26 November 2016 9,555 8 View

I'm looking for an online atlas ofthe human brain that allows input of xyz coordinates in MNI space to determine and label corresponding brain areas.

Can anyone point me in the right direction here please? many thanks

11 September 2013 6,106 11 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

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

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

Why is the efficiency of viral mRNA electroporation in PBMC low?

Hello, I have been struggling with the electroporation of PBMC with HIV-I mRNA. Although the quality of the RNA generated is good, and the handling of the cells in meticulous, the frequency of...

27 February 2021 9,233 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

Marcello Turi

Hi, I usually transfect even 3 plasmids together. Just split the total amount of DNA that you have in your protocol by the numbers of plasmids.

Suppose:

3 ug of total DNA

- 1.5 ug of luciferase plasmid

- 1.5 ug of his plasmid

Then add the amount of transfection reagent for 3 ug of DNA following your protocol.

Darius Vasco Köster

a little side note, for us, transfection with linear PEI instead works pretty well for HEK cells as an alternative to lipofectamine + electroporation. PEI is not very expensive and it allows you to transfect larger amounts of cells.