Is tracRNA essential for CRISPR in cell line expressing Cas9?

I have a cell line that stably expresses Cas9. However, I am not sure how to deliver the sgRNAs or gRNAs. Is it possible to buy tracRNA and ligate with sgRNA oligos before transfection? Or can I...

04 May 2019 6,418 5 View

Can I use exchange Stbl3 for MACH1 or DH5alpha cells?

I am using Dan Swarr and Dave Frank's protocol to ligate oligos to the PX459 vector, using Stbl3 cells (invitrogen). However, I get very few colonies (max 3) after transformation using 20ul of...

02 March 2019 8,761 2 View

How long is insulin and EGF stable in culture medium?

I am culturing primary cells and use DMEM F12 (1:1) cell culture medium supplemented with human insulin and EGF. How long are these components stable for in the medium? Thank you for your gentle...

02 March 2019 1,908 2 View

Can anyone suggest a protocol to isolate stomach epithelial cells for primary culture?

I want to isolate stomach epithelial cells, from both human and mouse, for primary culture. Does anyone has an easy to follow protocol withou resourcing to 3D cell culture?

09 October 2018 4,218 1 View

Is it possible to transport organs in PFA leak/spill-proof containers?

I need to transport embryonic organs fixed in 4% PFA and previously stained in b-gal solution (LacZ) to another country. Our objective is to embeed the tissues in paraffin. Is 4% PFA the best...

10 September 2018 4,794 3 View

Can we classify HV and LV lines based on line length?

I have dataset which shows the length of power lines. I need to classify the lines based on the line length. Is there a rule to classify the High voltage (HV) and low voltage (LV) lines based on...

03 March 2021 4,116 4 View

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02 March 2021 3,060 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 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

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 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

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 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

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