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Day Three Identification of Virus DNA in mixture of host cell DNA by antibody binding to DU labled Gemini specific probe Day three Detection of virus DNA Use DU specific antibody conjugate Page 196 steps 1-8 Record by digital photography step 9 10 Immuno-detection of virus specific probe From Last Year M I M I M=Not Infected I = Infected DS DNA SS DNA Week 3 TA will complete steps 12 page 414 Add 5ml of blocking buffer containing 1 microliter of antibody Incubate 30 min Pour into sink Wash twice with 1X washing solution Add 3 ml detection buffer Rock for 4 minutes Add 5ml of substrate Wait 30min. Check for detection Rx is complete in 5 hours This Lab Three weeks Report 40 points Pre Lab 9 points Next Week QPCR Lab D3 Page 253 Read introduction and Theory 264 Protocol page 266 VIRAL DNA PURIFICATION “Trino’s Lab” 231 FAMILY: Geminiviridae Genus: Begomovirus Species: Tomato golden mosaic virus (TGMV) Trino’s Lab This three week lab was conceived and implemented by Trino Ascencio as a graduate student in Biochemistry in the Lab of Dr. Linda Hanley-Bowdoin a world authority on plant DNA viruses. He is now Assistant Professor Properties of DNA Large polymers made up of nitrogenous bases and 5 carbon sugars linked by phosphodiester bonds. Nucleic acids because of phosphate groups with pKa of 1-2. Produces a strong acid with net – charge at neutral pH Average Amax is at A260 Amax Protein Properties of DNA BORING But…………………….. Relatively easy to Isolate Obtained in very high molecular weight form. DNA Molecule is designed to retain integrity mRNA by contrast intended to be degraded Protein subject to attack by enzymes and may be pH ionic strength and temperature sensitive Proteins have personality Different protocols are designed for different sources of DNA not differences in the molecule. GEMINIVIRAL DNA PURIFICATION GEMINIVIRUSES: Plant DNA viruses ssDNA (most plant viruses are RNA) Replication in nuclei Whitefly transmitted Problem worldwide GEMI
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