Repeated inserts ligated to my plasmid??

Hi all I ligated my small insert (37bp) into 3.5kb plasmid, I got colonies but when I check the sequence of my insert, I found more than one insert ligated as a repeats, some clone got three...

01 February 2019 4,050 6 View

How to get the exact number of the successful transformant bacteria???

07 August 2018 5,877 5 View

Anyone know a suicide plasmid that can be used to delete a gene in E.coli strain?

Anyone know a suicide plasmid that can be used to delete a gene in E.coli strain? I need to use a suicide plasmid to delete a gene in E.coli strain. I used one for gene deletion in Pseudomonas but...

11 December 2017 187 1 View

I found a mysterious thing after protein purification anyone can help?

Dear Dave I am working on a protein (around 125KD) and I have got some mysterious things regarding the purification so I need your help. The protein was expressed using pET28a vector in E.coli...

06 July 2015 6,863 18 View

How can I detect and measure the pyrophosphate PPi releasing from enzymatic reaction?

I have purified a protein which suppose to adenylate amino acid consuming ATP and releasing AMP and PPi, so to test the protein activity I have to measure the released PPi in the solution. Can...

08 September 2014 8,805 18 View

How can I add a DNA linker to a vector?

I am going to design two oligonucleotides (the size about 20 base) which can be annealed together forming two sticky ends then will be ligated to the digested vector with the corresponding enzyme....

03 April 2014 7,947 1 View

How can I chose the best pH for a protein solution?

For the protein to be stable and functional, which is the best pH of the buffer?

01 February 2014 5,410 0 View

Can someone help with flag-tag to the C-terminus of the protein?

Does anyone know how to add Flag-tag to the C-terminus of the protein? What is the principle of the procedure? I am planning to transform bacterial gene to mammalian cells and I have to check the...

31 December 2013 2,442 6 View

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02 March 2021 3,060 3 View

What element is driving the different slop in the low frequency tail?

My study consists simple corrosion inhibitors in salty brine. Right now I just fit my circuit with randles with CPE. Just wondering how the slop can given more info of the experiment. Or any...

01 March 2021 6,474 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

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 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

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 3 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