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Generation of a
stable
cell
line: A positive control plasmid works, but I cannot obtain
stable
clones with my construct of interest. What could be the reason?
One reason could be that your gene product is toxic to the
cells
. We recommend testing the construct for expression of the gene of interest by transient Nucleofection® of your
cells
. If the proportion of expressing
cells
drops between 4 and 48 hours...
Missing:
linesGuideline
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.
Missing:
linesGuideline
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
.
Missing:
linesGuideline
Is your pmaxGFP control vector supplied with the Nucleofector® Kits suitable for
stable
expression?
No. This vector is only supplied in our Nucleofector® Kits as a positive control and cannot be used for selection because it does not contain a mammalian selection marker. The only resistance gene expressed by this vector is kanamycin which is...
Missing:
cell
linesGuideline
How many
stable
clones will I get from one transfection?
This depends on the respective
cell
type (transfection efficiency, viability and integration frequency). If it is important to you to know the frequency of
stable
integrants before carrying out your actual experiments, the frequency should...
Missing:
linesGuideline
Why is the Nucleofector® Technology ideal for protein production from
stable
clones ?
The generation of a
stable
clone often requires six months or more due to selection procedures and adaption to serum-free conditions after transfection.With the help of the Nucleofector® Technology suspension
cells
can be transfected directly...
Missing:
linesGuideline
In
stable
cell
line generation, I have a very high transfection efficiency, but most of my
cell
s 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...
Missing:
linesGuideline
Following Nucleofection®, I cannot obtain
stable
clones from single
cell
s. What could be the reason?
Some
cell
lines need certain
cell
densities to proliferate. For those
cells
you can try using conditioned medium for setting up limiting dilution or culturing single
cells
together with feeder
cells
.
Missing:
linesGuideline
How long is the MycoAlert® Mycoplasma Detection Assay signal
stable
?
Please follow the protocol exactly as written – add reagent, wait 5 min and read, add substrate, wait 10 minutes and read again.
Missing:
cell
linesGuideline
What antibiotic concentration should I use for selecting
stable
transfectants?
We highly recommend to perform a titration for G418 in a range of 0,1 mg/ml to 1,5 mg/ml; in serum-free culture expand range down to 20 µg/ml).Choose the concentration which is 0,1 or 0,2 mg/ml above the one which shows complete
cell
death...
Missing:
linesGuideline
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