The stringency of hybridization in blotting and in PCR is dependent on physical parameters such as temperature and washing solution content. macromolecules ligation GMO lysis ligase transformation detergent plasmid transfection chelating agent transformation naked DNA EDTA competent vector nuclease electroporation electroporation pellet selectable marker microinjection PCR agarose vesicles primer vector callus thermalcycle band knock-out denature ethidium bromide recalcitrant anneal Southern blot Agrobacterium extend membrane particle Taq DNApol denaturation bombardment electrophoretic gel hybridization T-DNA restriction washing Ti plasmid endonuclease probe position effects EcoRI stringency stem cells sticky end northern blot blunt end western blot compatible end transgene 8.1 What information, and what EcoRI, which has only one site, reagents would you need to use PCR directly in the middle of the PCR to detect HIV in a blood sample? fragment? 8.2 A 6.0 kbp PCR fragment flanked c) What bands would be observed if by recognition sites for the HindIII the recombinant plasmid was cut with restriction enzyme is cut with HindIII both EcoRI and HindIII at the same then ligated with a 3kb plasmid time? vector that has also been cut with 8.3 If you started with 10 molecules HindIII. This recombinant plasmid is of double stranded DNA template, transformed into E. coli. From one what is the maximum number of colony a plasmid is prepared and molecules you would you have after digested with HindIII. 10 PCR cycles? a) When the product of the HindIII 8.4 What is present in a PCR tube at digestion is analyzed by gel the end of a successful amplification electrophoresis, what will be the size reaction? With this in mind, why do of the bands observed? you usually only see a single, sharp b) What bands would be observed if band on a gel when it is analyzed by the recombinant plasmid was cut with electrophoresis?
The stringency of hybridization in blotting and in PCR is dependent on physical parameters such as temperature and washing solution content. macromolecules lysis detergent chelating agent EDTA nuclease pellet PCR primer thermalcycle denature anneal extend Taq DNApol electrophoretic gel restriction endonuclease EcoRI sticky end blunt end compatible end ligation ligase plasmid transformation competent electroporation selectable marker agarose vector band ethidium bromide Southern blot membrane denaturation hybridization washing probe stringency northern blot western blot transgene GMO transformation transfection naked DNA vector electroporation microinjection vesicles callus knock-out recalcitrant Agrobacterium particle bombardment T-DNA Ti plasmid position effects stem cells 8.1 What information, and what reagents would you need to use PCR to detect HIV in a blood sample? 8.2 A 6.0 kbp PCR fragment flanked by recognition sites for the HindIII restriction enzyme is cut with HindIII then ligated with a 3kb plasmid vector that has also been cut with HindIII. This recombinant plasmid is transformed into E. coli. From one colony a plasmid is prepared and digested with HindIII. a) When the product of the HindIII digestion is analyzed by gel electrophoresis, what will be the size of the bands observed? b) What bands would be observed if the recombinant plasmid was cut with EcoRI, which has only one site, directly in the middle of the PCR fragment? c) What bands would be observed if the recombinant plasmid was cut with both EcoRI and HindIII at the same time? 8.3 If you started with 10 molecules of double stranded DNA template, what is the maximum number of molecules you would you have after 10 PCR cycles? 8.4 What is present in a PCR tube at the end of a successful amplification reaction? With this in mind, why do you usually only see a single, sharp band on a gel when it is analyzed by electrophoresis?