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Q1 – Do I need to check Impedance every time I run a pulsing protocol?
Yes, we recommend to always check Impedance by pressing the Ω button just prior to pulsing. This step will apply a very short and low amplitude AC trickle current that is used to get an accurate Impedance measurement. Please be aware that when performing the actual DC square-wave pulsing protocol, there is no measurement of Impedance at that time.
Q2 – Can I use the NEPA21 for bacterial or yeast electroporation-mediated transformations?
No, the NEPA21 is designed for complex low voltage pulsing patterns and is not suited for high voltage electropration required for bacterial and yeast cells. Please check out the ELEPO21 for this application.
Q3 – What is a “poring” pulse?
A “poring” pulse is a very brief, relatively mild electric field of mid-level voltage (in the 100’s of volts) designed to open holes in the plasma and nuclear membranes. Most brands of advanced electroporation devices offer only this single feature. See Q3, Q4 and Q5 to better understand the added features of the NEPA21.
Q4 – What is a “transfer” pulse?
A “transfer” pulse is a longer, low voltage field designed to transport negatively charged nucleic acid molecules into cells and nuclei. Only Nepa Gene’s technology allows for transfer pulses.
Q5 – Why reverse the polarity of the pulses?
Reversing the polarity of the transfer pulses increases the transfection efficiency for in vitro applications. Only the NEPA21 allows offers a polarity exchange feature.
Q6 – Why program multiple decaying pulses?
Like reversing the polarity, multiple decaying pulses increases the transfection effiency without further harm to the cell. This is one of the reasons that the NEPA 21 can deliver some of the best results in the industry.
Q7 – What electrode should I choose for my in vivo, in ovo, ex vivo experiments?
Provide us the details of your application and we will recommend the electrode types that have worked best for other researchers. Let Bulldog Bio be your personal consultant for these experiments.
Q8 – What type of electroporation cuvettes should I use with the NEPA21?
When using the CU500 cuvette adaptor, we recommend using conventional 2 mm gap electroporation cuvettes. Not all cuvettes are equal, and our data show that Bulldog Bio cuvettes provide equal or better results relative to more expensive brands.
Q9 – Do I need proprietary buffers for in vitro, adherent, primary cell transfections?
No, by using the NEPA21 efficient transfection of primary cells adhered to the bottom of cell culture plates can be successfully performed by using commonly available cell culture buffers.
Q10 – Why don’t you list the protocols for different cell and tissue types?
Because the electric pulsing is solely responsible for the transfection mechanism, different cell types require subtly different electrical patterns for optimal efficiency and toxicity balance. For this reason, we provide you with customized parameters upon request.
Q11 – How important is the quality of the DNA plasmid to efficient transfection efficiencies?
VERY! We find that it is necessary to use Endotoxin-Free Plasmid Purification Kits to ensure significantly better results.
Q12 – Is there a size limit for DNA plasmids that can be used in standard cell culture transfection?
Yes. We generally find that plasmids of 10kb or smaller work well with little modification from our standard protocols. Sizes above 10 kb do require modifications to the procedure and pulsing parameters. Contact us directly for more details.
Q13 – Can I use plasmid DNA for the TAKE or other transgenic zygote protocols?
No. Scientists have yet to develop a zygote electroporation procedure that is effective for plasmid DNA or double stranded DNA. Even long ssDNA (ODNs or LSSNs) can be very challenging. Contact us for specific advice.
Q14 – My Endotoxin-Free DNA plasmid kit gives plasmid concentrations far below your recommended levels. Can I still use it?
In general, we recommend that you first concentrate the DNA. Adding too much TE to an electroporation step can cause problems. Here are some guidelines for removing salts and concentrating DNA:
i) After eluting DNA from an endo-free kit, measure the concentration (via UV spec)
ii) Precipitate the DNA by adding isopropanol. Decant the supernatant and then aspirate it by using a tip
iii) After washing DNA pellet with 70% ethanol and centrifuging, once again decant and aspirate the supernatant
iv) Repeat the EtOH wash
v) Assume 100% recovery and add enough endotoxin-free TE buffer for a concentration of 10ug/ul. It is much easier to dilute to a lower concentration than needing to concentrate after the prep is completed.