How to detect biallelic mutation using T7 endonuclease assay?

I have transfected mouse ES cells with single gRNA, Cas9 system. I am able to detect cleaved bands after T7 endonuclease. However, how to determine if this is monoallelic or biallelic mutation?

11 December 2017 878 4 View

What should be the pulse length of electroporation for transfecting mammalian cells?

I am currently optimizing electroporation conditions for mouse embryonic stem cells. What should be ideal range of pulse duration?

06 July 2017 8,578 1 View

Has anyone used Biorad electroporation system to electroporate mouse ES cells E14Tg2a?

I wish to know electroporation conditions especially for transfecting Cas9 RNA and protein

11 December 2016 2,484 2 View

Is it a good idea to do an overlap extension PCR with KAPA polymerase?

I have tried amplifying my individual fragments using Phusion but it is not working consistently but always works fantastically with KAPA. I think I am unnecessarily wasting time on Phusion....

05 June 2016 9,302 4 View

What should be the ideal ratio of primer to template DNA in PCR?

I have tried doing a PCR with kapa as well as Phusion HF polymerase. I am getting amplification with kapa, but Phusion is just not working. Both the master mixes use same: 1.5mM MgCl2 and 0.2mM...

04 May 2016 6,915 3 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

Isolates of Hymenoscyphus fraxineus?

Dear Dr. Cai, my name is Simone Prospero and I work in the team of Phytopathology at the Swiss Federal Institute for Forest Snow and Landscape Research (WSL;...

01 March 2021 1,133 1 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

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

How to eliminate Steady state error from Reinforcement learning genetic algorithm code?

The following code (see 1st 2 images attached) is used to produce PID controller values that are designed to control the system (G). The code finds the PID controller values (noted as k) by using...

28 February 2021 6,560 14 View

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28 February 2021 9,936 2 View