Name:________________________________________ TA:____________________ 8 Question 4 (28 points) You are studying a small experimental plant known as Cactusus experimentalis. You would like to create a genomic library of Cactusus experimentalis so you can find the gene responsible for touch sensitivity, called TOU. a) List the three features your cloning vector must contain to be useful in construction of a genomic library. 1. 2. 3. b) After isolating the genomic DNA, what enzymes and reagents would you need to buy for the remaining steps of constructing a genomic library? (Circle ALL that apply.) Enzymes Reagents DNA Polymerase dATP, dCTP, dGTP, dTTP RNA Polymerase ddATP, ddCTP, ddGTP, ddTTP Ligase Primers Restriction enzyme E. Coli Reverse Transcriptase Human Cells Cloning Vector DNA template ATP, CTP, GTP, TTP You create the genomic library (starting with an EcoRI digesting of the DNA), and identify one vector that contains your TOU gene. You decide to analyze this vector further. You cut the vector with EcoRI and purify the genomic insert. You then digest the insert with 2 different restriction enzymes SpaII and HindIII. You obtain the following results:Name:________________________________________ TA:____________________ 9 Question 4, continued Uncut HindIII SpaII HindIII Marker insert & SpaII c) Draw a map of the genomic insert indicating restriction sites for the enzymes SpaII and HindIII. EcoRI sites are already shown for you. Be sure to include distances. EcoRI EcoRI You then decide to sequence a small portion of the TOU gene found on your insert. d) Circle ALL the enzymes and reagents on the following list that you would need to perform a sequencing experiment. Enzymes Reagents DNA Polymerase dATP, dCTP, dGTP, dTTP RNA Polymerase ddATP, ddCTP, ddGTP, ddTTP Ligase Primers Restriction enzyme E. Coli Reverse Transcriptase Human Cells TOU gene insert ATP, CTP, GTP, TTPName:________________________________________ TA:____________________ 10 Question 4, continued You obtain the following gel after amplification. e) What is the sequence represented on the gel? Be sure to indicate the 5’ and 3’ ends. f) After sequencing the TOU gene obtained from your genomic library you find a 32 base pair insert in the middle of it that does not correspond to the mRNA for TOU. What is it? g) You want to express the TOU gene in E. coli. i. Given the result in part f, would you want to use genomic or cDNA library for this experiment? Why? ii. What organism must the promoter be from?
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