How can I transform 50 kb plasmid into ecoli?

I must put 50 kb plasmid into ecoli strain. I think 50 kb is too big for heat shock with ecoli. Is that correct? Will electroporation work? Or do I need to use cos sites and lambda phage...

22 January 2021 8,612 3 View

Bacteria strain received has fungal contamination -- how remove?

I received several strains of bacteria from another lab far away. However, one of the samples appears to have high fungal contamination. When I grow it in broth, I see lots of fungal...

03 January 2021 2,749 16 View

New knockout bacterial strain: Can't electroporate it?

I made a new bacterial strain with an unmarked deletion using 2 step homologous recombination (with sacB counterselection). The full protocol is given in the link at the bottom. The protocol...

01 January 2021 1,580 4 View

Western blot: what causes patchy, uneven background?

Sometimes my western blots have very uniform/even background, other times they're very uneven and patchy like in the attached image. Why is this? I thought my blots were drying out during...

05 December 2020 8,656 5 View

Licor Intercept Blocking Buffer -- sterility and reusing antibody dilutions?

In the past, I've always used Licor's old blocking buffer that included sodium azide. I would dilute my antibodies in the buffer, stain, re-collect the dilution, and re-use it a few times. The...

28 November 2020 5,795 3 View

What mutations in oriE increase the plasmid copy number?

I have a low copy plasmid (ori=pBR322) and I wanted change it to high copy (like ori=pUC). This paper seemed to say that only one mutation was required to increase the copy number: High copy...

13 November 2020 7,292 4 View

Western blot: adjusting volume of buffer for blocking/staining?

For western blots, I normally stain with 10 mL buffer with 1:1,000 dilution of my specific antibody. Recently, there is an equipment change in my lab and 10 mL isn't enough volume to cover the...

27 October 2020 2,749 7 View

Is this correct molecular docking?

Hello, buds I need your help to interpret the attached data. R group in serine is CH2OH, but the data show the two O in the group with hydrogen bonds with a molecule. Is this correct?...

02 October 2020 7,787 6 View

Concentrating protein: comparison of methods?

I must concentrate and purify a lot of dirty lysate and secreted protein samples. I just want to run them on protein gels for western blots. I'm trying to decide the best method of...

10 September 2020 2,911 8 View

Oxygen detection using Ru(bpy)?

I am trying to detect if my nanoparticles generate oxygen. Is there a way I can use Ru(bpy) and obtain an emission spectra? (Any other "quick and fast" method I can use to detect oxygen generation?)

26 August 2020 1,942 3 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 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

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

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

What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View

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28 February 2021 9,936 2 View