IPSC differentiate after thawing, can anyone help troubleshoot please?

Dear Colleagues, I am having problems with resuscitating cryopreserved iPSCs. Upon thawing I get quite a good survival rate, but all my colonies look completely flat, without smooth borders and...

03 April 2016 6,524 7 View

Is it possible to measure mitochondria number in cells by super resolution microscopy (STORM) using a good mitochondrial fluorescent marker?

Hello All, I would like to ask if anyone know if measuring mitochondria number in cells could be feasible by using STORM microscopy to detect a specific fluorescent mitochondrial marker. If yes is...

10 November 2015 272 5 View

How important is the orientation of a transgene when using CRISPR to knock-in a cassette in a safe harbour locus?

Hello, I am trying to use CRISPR/Cas9 for the knock-in of a transgene using HDR by targeting the safe harbour locus AAVS1 in human iPSC.Is orientation of the transgene important for its...

08 September 2015 6,717 3 View

For transgenesis is it better to knock-in a cDNA sequence or a whole gene?

08 September 2015 7,974 4 View

Mitomycin C precipitates in PBS solution after storing a -20 degrees. Is this deleterious to cells/compound?

Hi everyone, I have recently purchased mitomycin C from SantaCruz to inactivate MEFs and since insufficient information was provided by the manufacturer on reconstitution and stability I had a...

01 February 2015 2,911 6 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

Can NFL theorem be valid in infinite search space in ML?

NFL theorem is valid for algorithms training in fixed training set. However, the general characteristic of algorithms in expanded or open dataset has not been proved yet. Could you show your...

01 March 2021 1,189 3 View

Is there any research regarding how training and development is going to change in the era of Fourth Industrial Revolution?

As the organizations getting prepared for the fourth industrial revolution, to enhance the skills of their workforce what type of changes are necessary and going to occur in the training and...

01 March 2021 3,199 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

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

Which group formation theory do you recommend for projects that aim to bring together entrepreneurs for training for regional development?

I would like to have ideas about which theories of group formation are suitable for entrepreneurial training projects, with the aim of creating bonds, building trust, and collaboration for...

28 February 2021 4,223 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

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

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