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Le Virus de l'Immunodficience Acquise (HIV)-1 reste un problème majeur de santé publique dans le monde. Malgré des thérapies antivirales efficaces permettant d'allonger la durée de vie des patients infectés par HIV-1, aucun vaccin capable de protéger de l'acquisition du virus ou curer l’infection n'a été homologué. Nous avons développé un vaccin le...
HIV-1 remains a major public health issue worldwide in spite of efficacious antiviral therapies, but with no cure or preventive vaccine. The latter has been very challenging, as virus infection is associated with numerous escape mechanisms from host specific immunity and the correlates of protection remain incompletely understood. We have developed...
I'm working on the immunogenicity of a DNA vaccine. To do so, I plan to inject the plasmid in mice by Intradermal route + Electroporation in the back skin, with forceps-like electrodes.
As it is the first time I'm using electroporation device, I have few questions :
- Do I need to calculate the distance between the electrodes to define the voltage setting on the machine ? e.g. I want to use 100V/cm : should I set directly 100V on the machine or should 1) measure the distance between the electrode (e.g. 7mm) ; and then 2) calculate the correct voltage (70 V) ?
- Did somebody experienced the CUY21 edit II electroporator (BEX CO. LTD) ?
-Also, do you recommend to use conductive gel ? I saw the Highly Conductive electrode gel (Signa gel - Parker Laboratories), can I use it on the electrode ?
Finally, I've read that a combination of high + low voltages are better for the DNA to penetrate into the cells, but I'm afraid to damage the skin... Any suggestions ?
Any help and advises are appreciated ! :)
Thanks a million,
I've transformed high-copy plasmid (pGEMT Easy - 13 Kbp) in E.coli (JM109) and did plasmid extraction with commercial kit from Macherey Nagel GigaPrep EF. However, the resulted concentration of DNA is very low, of about 1-2 mg instead about 10 mg ...
I used 2L LB media with kanamycin (4*500mL in 2L bottles for a better O2 exchange) at 32 degrees C, 200rpm for about 20h.
I weighted the bacteria pellet after culture, it was around 9g for 1 L of bacterial culture (should be between 3-20g/L for high-copy plasmid according to the manufacturer)
I proceed the extraction as mentioned by the kit protocol.
Any idea how to get better DNA concentration?
Thank you for your help !