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MACS Selection Kits (Miltenyi Biotec GmbH) offer the opportunity to purify specific blood/immune cell populations by positive selection via antibody-binding or by negative selection using depletion. Which MACS purification method is preferred by Lonza?

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

I've noticed that duration of the Nucleofector Program seems to change even when I haven't changed the program I am using. Why?

The duration time of a program is dependent on the number of the program. The duration is also dependent on how long the Nucleofector™ Device has been turned on as well as the number of Nucleofections you have done in the last couple of minutes. When...

Is it typical for the fluorescence observed from my tagged protein to appear less than the empty fluorescent vector?

Yes, this is a routine observation. Fluorescent fusion proteins can appear less bright than the fluorescent protein itself. This may be due to differences in protein folding of the fused protein.

What is the lowest amount of DNA which can be used for transfection? Do I need a DNA carrier for low DNA amounts?

The recommended amount of DNA per 100 µl reaction is 0.5µg-5 µg. No carrier is needed.

Lonza recommends using PEG purified DNA for Nucleofection of DC's and macrophages. Is it necessary to perform the PEG purification step for the positive control plasmid pmaxGFP supplied in each Nucleofector Kit?

No, it is not. The PEG purification step removes additional endotoxin from the DNA preparation. The pmaxGFP reporter plasmid supplied in each kit is highly purified and endotoxin levels are <0.01E.U./µg DNA (which means <1pg endotoxin/µg DNA).

How critical is the time point for analysis post transfection?

It is very critical. Waiting too long for analysis post transfection can lead to loss of peak expression. The optimal time for analysis is related to two factors: stability of protein being expressed and transfection method. For a short-lived...

Can I use larger cuvettes for my Nucleofection Reaction ? Can I use the cuvettes more than once?

No. The electrical parameters provided by the Nucleofector™ Device are optimized for the cuvettes contained in the Nucleofector™ Kits. The cuvettes are single-use only. Using the cuvettes more than once will result in higher cell death and lower...

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.

Do we sell the Nucleofector Solution (or any other component of the Nucleofector Kit) separately?

We don't sell the individual components of the kits separately because our product line is a kit concept line to ensure the use of Amaxa™ certified products with quality controlled lot numbers that guarantee optimal results.

Can nicked DNA lead to reduced transfection efficiency?

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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