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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).

Can mycoplasma grow in the absence of cells?

There is evidence to suggest that mycoplasma can survive in the absence of cells, some may even proliferate, but as a rule they are much more viable if cells are present. 

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...

Is it possible to store reconstituted MycoAlert®/MycoAlert® PLUS Reagent and Substrate?

For optimal assay conditions, reconstituted reagent and substrate should be used fresh within 2 hours after reconstitution.During this time they can be kept at room temperature.For the MycoAlert® Kit: Unused, reconstituted components can be aliquoted...

Can I use a medium other than the one you offer to culture Clonetics® Cells?

We have an optimized medium for each Clonetics® Cell type. The cells are grown and tested in their specific medium system. Lonza guarantees the performance of Clonetics® cells only if appropriate Clonetics® media and reagents are used...

DNA-purity and Nucleofection®: Can low A260:A280 ratios lead to both reduced transfection efficiency and cell viability?

Yes. To check the quality of your DNA we strongly recommend determining the A260:A280 ratio. It should be ideally 1.8 - 2.0 for a good DNA preparation, but least 1.6.

What kit sizes are available for the 4D-Nucleofector® X Unit?

We offer two different kit sizes for the 100 µl Nucleocuvette® Format (12 and 24 reactions) and one size for the 20 µl 16-well Nucleocuvette® Strip Format (32 reactions). 

How much sample do you need for accurate results with MycoAlert® Assay? Do you need cells or just supernatant?

The standard protocol calls for taking 2 ml of culture media, spinning down any cells and removing 100 µl of supernatant as sample.Smaller initial aliquots of media may be taken to start with if necessary.

What cell numbers can I transfect when using the 4D-Nucleofector® System?

The 4D-Nucleofector® System offers great flexibility in terms of cell numbers.Using the 4D-Nucleofector® X Unit it is possible to perform Nucleofection® in 100 µl and 20 µl Format.The 100 µl Nucleocuvette® Format is suited for high cell...

Can nicked DNA lead to reduced transfection efficiency in Nucleofection® experiments?

Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded. Compare undigested plasmid to plasmid DNA digested with a suitable single cut restriction enzyme to linearize. Supercoiled plasmid will run faster than...
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