By Monica Rinaldi, Daniela Fioretti, Sandra Iurescia
DNA Vaccines: tools and Protocols, 3rd version explores leading edge methods and applied sciences used to layout, convey, and improve the efficacy of DNA vaccines. that includes purposes which could be of serious price in relocating vaccines from learn to hospital, this specified quantity contains sections on DNA vaccine layout and enhancement, supply platforms, construction, purification, and caliber, in addition to chapters on new vaccine purposes. Written within the hugely profitable Methods in Molecular Biology sequence structure, chapters comprise introductions their respective issues, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and pointers on troubleshooting and heading off recognized pitfalls.
Authoritative and functional, DNA Vaccines: equipment and Protocols, 3rd Edition serves the $64000 function of extra documenting the opportunity of the DNA vaccination as a platform expertise for therapy and prevention of human disease.
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Extra resources for DNA Vaccines: Methods and Protocols
Orbital shaker, 37 °C. 2 Transformation of E. coli DH5α Cells 1. Competent E. coli DH5α cells. 2. Plasmids: pVAX1-N3-GFP (N3 vector), pVAX1-E1A-N3LAMP (E1A-N3-LAMP), and pVAX1-ISG-GFP-LAMP (ISGLAMP), and pVAX1-Sc-ISG-GFP (Sc-ISG) encoding for the invariant surface glycoprotein (see Note 1). 3. Sterile liquid LB medium (25 g/L). 4. LB-agar plates (35 g/L) supplemented with kanamycin (30 μg/mL). 5. Water bath. 6. Incubator, 37 °C. 7. Orbital shaker, 37 °C. 8. Cryovials. 9. Glycerol for molecular biology (99 %).
After electrophoresis, remove the gel from the electrophoresis chamber and cut it longitudinally between wells two and five. The gel portion containing the molecular weight ladder and the 2 μL sample is then removed from the tray and stained for 20 min in an ethidium bromide solution. Protect the second portion of the gel by placing aluminum foil under it. 16. Put on the UV protection mask, and with a scalpel tip, mark the bands corresponding to the vector pVAX1-N3-GFP and to the different insert targeting sequences on the stained portion of the gel.
Scalpel tip. 14. Transilluminator. 15. QIAquick Gel Extraction Kit (QIAGEN). 16. 80–100 % (v/v) isopropanol. 17. Preheated sterilized MilliQ water at 65 °C (see Note 4). 18. Spectrophotometer. 38 Elisabete Borges Freitas et al. 6 Ligation and Bacterial Cell Transformation Processes 1. Fragment corresponding to the linearized vector pVAX1-N3GFP and the inserts (E1A, LAMP, or Sc). 2. T4 DNA ligase 3 U/μL. 3. 8, 100 mM MgCl2, 100 mM DTT, 10 mM ATP. 4. MilliQ sterilized water. 5. DNA SpeedVac® Concentrator.
DNA Vaccines: Methods and Protocols by Monica Rinaldi, Daniela Fioretti, Sandra Iurescia