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How long does it take to obtain stable transfectants?

Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition.

What does the message "weak" mean on the display of the Nucleofector® IIN, IIS or 2b after Nucleofection®?

This message means that the system cut off the pulse before it was completed (usually this is because the conductivity of the solution is at the low end of the normal range). The pulse was applied; it is just going to be a little weaker than...

In stable cell line generation, I have a very high transfection efficiency, but most of my cells die during selection. Is this to be expected?

This is the normal pattern you should expect to see. Only a small proportion (1/10,000 to 1/100) of all transfected cells will integrate the transfected DNA into their genome and become stable transfectants. The remaining cells lose the transfected...

Where can I find the meaning of the 4D-Nucleofector® System Error Codes?

In the back of the respective manuals, there is a chart giving a brief description of the error codes. If more information is needed or an error code is recurrent, it is best to contact Scientific Support for advice to determine if there is a...

What selection markers can I use for the generation of stable cell lines?

The most commonly used marker is the neomycin phosphotransferase (neo) gene that confers resistance to G418 to eukaryotic cells. Other markers are Puromycin, Hygromycin, Zeocin, or the HPRT gene that can be used in HPRT-deficient cells.

What are useful methods to enrich/purify specific hematopoetic cell populations before Nucleofection?

Specific blood cell populations can be enriched/purified for Nucleofection™ by MACS™ selection Kits (Miltenyi Biotec GmbH), RosetteSep™ Kits (StemCell Technologies Inc.) or Dynal™ beads (Invitrogen Corporation).

At which time point after Nucleofection® should I start selection of stable integration?

Antibiotics for selection should be added 24-48 h after Nucleofection®.

Do you recommend a purification method to purify specific blood/immune cell populations before Nucleofection®?

We recommend using negative selection (depletion) because your purified cells are "untouched", not influenced, and strictly not activated.Positive selection may cause increasing amounts of dead cells and could also lead to activation of the cells due...

According to which standard is the Nucleofector® System certified?

The 4D-Nucleofector® Unit was tested by TÜV Rheinland Product Safety GmbH and found to be in compliance with the following standards:• IEC 61010-1The 4D-Nucleofector® Unit was tested by TÜV Rheinland of North America Inc. and found to be in...

How often should I change the medium during selection of stable clones after Nucleofection®?

The culture medium should be changed every 2-3 days during the selection process.This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.
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