Clarity Software for Biotek Microplate Luminometer?

My advisor and I recently extracted high-molecular weight DNA from Acanthamoeba polyphaga using Cells and Tissue DNA Isolation Kit (Magnetic Bead System) by Norgen Biotek Corp. Our objective is to use the HMW DNA for PacBio Sequencing.

Today, we ran the DNA on a 0.7% agarose gel (stained with Sybr Safe). We are baffled by the the bright bands toward the top of the gel in the second and third lane from the left (that's the Acanthamoeba DNA). If the DNA were sheared, I would think that there would be a smear over the entire lane. Instead, there are two distinct bands with DNA that is <250 nucleotides long and DNA that is about 48k nucleotides long. The bands in lanes 4-9 are Lambda Phage DNA.

What could those short bands be from? The band at the top of lane two is also slightly more yellow than the rest. Is there some sort of size selection method we can use to obtain the larger fragments of DNA?

Hello everyone,

I am using mitotracker green to evaluate the total number of the mitochondria in primary cells after stimulation with different materials and i used Cytation 5 to measure the fluorescence intensity by area scanning, i used 48 well plate but several experiments i noticed the that the wells located in the middle section of the plate have always lower number comparing to the wells on the peripheral even for the same sample. Does any one face this before? or can any one tell me a valid way to quantify the mitochondrial number not using microscope?

I really appreciate your help in this.

Hello, I bought a Micro-plate reader 800 Ts Biotek, it has a Gene5 3.11 software. However, I want to use it for Diagnostic ELISA and I've already have the standard factor, so I don't need to create a Standard Curve. I was wondering if I can use the Software to analyses automatically typing the standard factor and I'll obtain the Index.

Regards,

Rita

I am trying to using the Gen5 Cytation by BioTek for protein quantification using a micro BCA Assay kit.

I downloaded a BCA Assay Experiment (.xpt) File, and tried to use it but ran into some trouble. When setting up/entering info for the plate map, I was able to add the BSA standard wells with no problem because I knew the concentrations of each standard in ug/mL.

For unknowns it didn't let me specify my own dilutions. There were some options given (1:1, 1:2, up to 1:96) but it wouldn't let me choose the dilutions that I had since my dilutions didn't start at 1:1 and mine increased in larger increments.

Am I stuck using those dilutions suggested by Gen5 or does someone know a way to set up the protocol differently?

I am looking for one that prints de result in the equipment by itself and also has the possibility to export all produced data to a computer.

Thank you in advance.

For telomere elongation, 2 μL of telomerase extract with various concentrations ranging from 101 to 106 cells was incubated with 1 μL TS primer (100pmol) in 10 μL reaction buffer (50mM Tris-HCl, 150mM NaCl, 10mM, KCl, 1mM MgCl2, pH 7.4) at 37 °C for 1 h.

I also need dNTPs for this reaction but I am confused about the volume of dNTPs needed? ( dNTPs 1mM)

Hi! Recently we started using Cytation 1 in our lab to count cells and see them on a plate. I have no problems counting HEK 293T cells in 24 well plates from "Greiner bio-one" or "Thermo Nunclon Delta Surface" (look at them, they look so pretty!). But when we move on in a 48 well plate from the same brands, Cytation 1 cannot focus, and we have results like the ones in the second picture.

Also, my colleagues are having problems with the 24 well plate but counting fibroblast instead of HEK. We didn't change a thing in the settings, and we are really mad about it. Any ideas?

Thanks!

Hi,

we are trying to set up the qEV columns for EV isolation and we are experiencing difficulties with reproducibility. Prior to loading the EVs we concentrate using Amicon filters. Once we have all the fractions we run analyze them by western blot for the presence of CD63. However, we found that CD63 is always present in a completely different fractions and we also realized that the EVs are already going through the column during the first washing step (as these fractions are positive for multiple EV markers). We already tested different columns and we cannot find the solution why EVs would be going through the column so quickly. Does anyone have any idea? Any advice would be greatly appreciated!