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How much DNA can I use when transfecting hepatocytes with the standard Nucleofector™?

We have used up to 6 µg of DNA per 100 µl reaction with no deleterious effect.

For hepatocytes, is plating density a concern post Nucleofection™?

Yes, the optimized protocols for the standard Nucleofector™ recommend the use of 6 well plates. Plating into a 12-well plate will pose a problem as less cells will attach at high densities.

Four hours after Nucleofection™, I can see the hepatocytes have attached to the well, but the morphology does not look correct, should I be concerned?

There is no reason for alarm. The hepatocytes may not exhibit normal morphology a few hours after Nucleofection™ but by 24 hours post Nucleofection™, morphology should be normal. Remember to perform a fluid change about 4 hours post Nucleofection™....

Does the 96-well Shuttle™ recognize if the cuvette plate is in backwards?

No, it does not recognize the orientation of the plate. Please make sure that well A1 is positioned in the upper left corner of the retainer.

Which reporter gene should I choose for my reporter gene experiment?

In general, all reporter genes can be used. For some applications (e.g. reporter gene experiments in various primary cells, experiments with late analysis time point due to starving and /or stimulation) a reporter gene with a longer half life than...

Do any of the three instruments used in the 96-well Shuttle™ System "go to sleep" or power off automatically?

The energy options of the laptop are set as such that only the screensaver is activated, the harddisk won't turn down and hibernation is disabled. One can change these settings via the system control. However this is not recommended because by...

What is the maximum amount of DNA I can use in my Nucleofection™ Experiment?

The overall amount of 5 µg should not be exceeded. Look in the cell specific Optimized Protocol (OP) for the recommended total DNA amount of the cell type of interest.

What is the molecular weight of your control pmaxGFP™ plasmid?

The molecular weight is approximately 2.3x10e6 g/mol based on the average MW of a base pair being 660 g/mol.

When should I begin looking for the Luciferase signal after Nucleofection™?

The time course of plasmid expression after Nucleofection™ might be different from the time course seen with other transfection methods. We recommend looking at different time points and start with the first time point as early as 4 to 6 hours post...

What should I pay attention to when transferring my reporter gene set up from a cell line to a primary cell?

In general the overall expression level differs between different cell types. Don't expect the same level of gene induction when working with primary cells. You might need to change to other DNA amounts or ratios.

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