By S. Moira Brown, Alasdair R. MacLean
A good choice of state of the art experimental equipment for herpes virology. those tactics diversity from the extra organic in vivo maneuvers to the merely molecular, and are defined intimately through chosen specialists to make sure reproducibility. many of the protocols depend upon the herpes simplex virus as prototype, in order that it then turns into really effortless to extrapolate and make the mandatory adjustments required for program to different herpesviruses, specifically individuals of the alpha team, akin to PRV and EHV.
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Extra info for Herpes Simplex Virus Protocols
19 Other requirements-enzymes* restrtctton enzymes, Calf Intestmal Phosphatase (CIP), polynucleottde kmase, T4 DNA polymerase, T4 DNA hgase. Nowadays, enzymes generally come with the appropriate reaction buffers. A neutralized solution containing each of the four dNTPs at a 2-mM concentration. , Boehringer or Stratagene. A phage lambda packaging kit A Geneclean (Bio 101) or similar ktt for elutton of DNA fragments from agarose gels. 1-mL syrmges, 25-gage needles. 5 x 2-m ultracentrtfuge tubes Electroelution equtpment.
8. 1 and 1 pg of lambda DNA to estimate its concentration. 4. Addition of Synthetic Linkers to the Sheared DNA Fragments and to the Cosmid Vector The ends of the sheared- and size-selected vnus DNA fragments are blunted, after which synthetic linkers are to be added at the ends for clomng (see Note 10). , a unrque BumHI site) de Wind, van Ziji, and Berns 56 must be converted to the new unique cloning site by linker addition, for which we also provide the protocol. 5. 3. 5. Blunting the Sheared Virus DNA Fragments and Addition of a Restrict/on Site for Cloning This subprotocol mcludes a number of separate enzymatic steps: 1.
5 ng), 2 pL ligation buffer, 5 (Weiss) Umts T4 ltgase, distilled water to 20 pL. Incubate overnight at 15°C. 5 Add to each tube 80 pL TE and 100 pL phenol chlorofonnlsoamyl alcohol, vortex gently for 30 s, centrifuge for 2 min at maximum speed, and transfer the supematant to a clean micromge tube. Subsequently, prectpnate the DNA by additton of 0 1 vol3M sodium acetate pH 5 2 and 2 vol ethanol Store on Ice for 15 mm and centrtmge for 5 mm at maximum speed Wash the pellet with 1 mL 70% ethanol and dtssolve the DNA m 10 pL TE by heating for 15 min at 65’C or by overnight incubation at 4°C.