Antiviral Methods and Protocols by Peggy Geluykens, Koen Van Acker, Johan Vingerhoets, Christel PDF


By Peggy Geluykens, Koen Van Acker, Johan Vingerhoets, Christel Van den Eynde (auth.), Edwin Yunhao Gong (eds.)

ISBN-10: 1627034838

ISBN-13: 9781627034838

Although antiviral medications were effectively constructed for a few viral illnesses, there continues to be a transparent, unmet scientific have to boost novel antiviral brokers for the keep watch over and administration of many viruses that presently haven't any or constrained treatments in addition to a necessity to beat the restrictions linked to the prevailing antiviral medications, akin to antagonistic results and emergence of drug-resistant mutations. the second one version of Antiviral equipment and Protocols features:

  • All chapters are new and written through specialists within the box, reflecting the main contemporary technical advances in antiviral study and discovery.
  • This variation makes a speciality of many very important human viruses, corresponding to human immunodeficiency virus variety 1 (HIV-1), hepatitis viruses (hepatitis B and C viruses), herpes viruses, human breathing syncytial virus (RSV), and influenza virus, whereas additionally that includes a few vital rising viruses, equivalent to dengue virus, West Nile virus, and chikungunya virus.
  • As a quantity within the hugely winning Methods in Molecular Biology sequence, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and pointers on troubleshooting and fending off recognized pitfalls.

Comprehensive and state of the art, Antiviral equipment and Protocols, moment Edition will function a great laboratory reference for pharmaceutical and educational biologists, medicinal chemists, and pharmacologists in addition to for virologists within the box of antiviral study and drug discovery.

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Thermocycler: Gene Amp PCR System 9700 (PerkinElmer). 3 Amplicon Purification and Gel Analysis 1. QIAquick 96 PCR purification kit containing the extraction buffers PE, PM, and EB (Qiagen, Cat. No. 963141). 2. QIAVAC 96 well (Qiagen, Cat. No. 9014579). 3. V bottomed 96 well plates and cover (Costar). 4. Absolute ethanol. 5. 3 (Invitrogen). 6. 6× DNA loading dye. 7. Generuler™ 1 kb Plus DNA ladder (Fermentas, Cat. No. SM1333). 8. 5 μg/mL ethidium bromide (Lonza, Cat. No. 57234). 42 Peter Van den Eede et al.

2. 0), 1 mL of 1 M MgCl2, 159 mL of UltraPure DNase/RNase-free distilled water. 3. Sequencing primers (see Tables 3 and 4). 5 Purification of Sequencing Reactions and Capillary Gel Electrophoresis 1. ABgene Thermofast 96 PCR plate. 2. DyeEx kit (Qiagen, Cat. No. 63183). 3. Clearseal 3730. Genotyping of the HIV-1 PR- RT and IN Genes 43 Table 3 HIV-1 PR–RT sequencing primers Name Sequence (5′ to 3′) Position of Concentration working primers HXB2 solution (μM) F1 GAGAGCTTCAGGTTTGGGG 2170–2188 4 F2 AATTGGGCCTGAAAATCC 2696–2713 4 F3 CCTCCATTCCTTTGGATGGG 3222–3241 4 F5 CACTCTTTGGCAACGACCC 2261–2279 4 R1 CTCCCACTCAGGAATCC 3778–3794 4 R3 CTTCCCAGAAGTCTTGAGTTC 2797–2817 4 R5 GGGTCATAATACACTCCATG 3492–3511 4 R6 GGAATATTGCTGGTGATCC 3012–3030 4 F4 CAGACCAGAGCCAACAGCCCC 2142–2162 4 F6 GGTACAGTATTAGTAGGACC 2469–2488 4 F7 GTACTGGATGTGGGTGATGC 2871–2890 4 F8 GTGGGAAAATTGAATTGGG 3330–3348 4 R2 GTACTGTCCATTTATCAGG 3255–3273 4 R4 CTAACTGGTACCATAATTTCACTAAGGGAGG 3807–3837 4 R7 CATTGTTTAACTTTTGGGCC 2601–2620 4 R8 GATAAAACCTCCAATTCC 2397–2414 4 The first eight primers are the first-line sequencing primers and the last eight primers are the backup sequencing primers Table 4 HIV-1 RT–IN sequencing primers Name Sequence (5′ to 3′) Position of primers HXB2 Concentration working solution (μM) F3771 GCCACCTGGATTCCTGAGTG 3771–3790 4 IN_F_4074 CAACCAGATAAAAGTGAATCAG 4074–4095 4 IN_F_4540 TAGCAGGAAGATGGCCAGT 4540–4558 4 IN_R_4348 CTCCTTTTAGCTGACATTTATCAC 4348–4371 4 Inseq2R CTGCCATTTGTACTGCTGTC 4748–4767 4 VIF_R_5193 ATGTGTACTTCTGAACTT 5193–5210 4 44 Peter Van den Eede et al.

Create the donor DNA by annealing oligo INdon1 and oligo INdon2 to each other by adding 25 μL of INdon1 stock solution to 25 μL of INdon2 stock solution in a 2 mL Eppendorf tube (Fig. 2), and then add 450 μL of annealing buffer. For annealing of the oligos, preheat the heating block to 95 °C, then remove the blocks from the apparatus, place the tubes in the block, and let them cool down inside the block for 30 min. Store the tubes at −20 °C. 1. 2, step 1): Prepare 50 mL assay buffer per 384-well compound plate.

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Antiviral Methods and Protocols by Peggy Geluykens, Koen Van Acker, Johan Vingerhoets, Christel Van den Eynde (auth.), Edwin Yunhao Gong (eds.)

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