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Describe the gases (including oxygen and carbon dioxide partial pressure percentage ranges), nutrients, and waste products. Explain the relationship between RBC antigens and ABO blood group types. Identify the stages and processes involved in the clotting processes of blood. ESSENTIALS OF ANATOMY AND PHYSIOLOGY (8th edition)

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III. DNA techniques (8pts): You are a researcher working in a lab that studies "uncharacterized" mystery proteins. You have identified a hypothetical protein in the genome of Bacillus subtilis and want to express this protein so you can crystalize it and determine its structure/function. Below is the coding sequence of your gene (ATG = translational start codon, TAA = translational stop codon. These set the boundaries for your coding sequence): 5'- ATGGCTAACCAAAACTCTTCAAATGACTTACTAGTTCCTGGCGCAGCTCAGGCTAT CGATCAAATGAAACTTGAAATCGCTTCTGAATTCGGCGTTAACCTTGGAGCGGACA CAACTTCTCGCGCTAACGGTTCTGTCGGAGGAGAAATCACAAAACGTTTAGTATCT TTCGCTCAGCAGCAAATGGGCGGCAGAGTTCAATAA-3' A) Design a pair of primers that will amplify this gene (20 bases each). Make sure you indicate the directionality of your sequence (2pts). Forward: Reverse: B) You need to clone this gene into a vector and decide to use the restriction sites Bglll (5'-AGATCT-3') and EcoRI (5'-GAATTC 3'). You need the Bglll site to be at the beginning of your gene sequence, and the EcoRI sequence to be at the end. Where do you add this sequence on your primers (2pts)? Forward: Reverse: C) You amplify this gene with PCR, double digest it with BgIII/EcoRI enzymes and then clone it into a vector also digested with BgIII and EcoRI. Next, you must validate your construct (the vector/plasmid you created) through restriction digestion using other restriction enzymes. You digest it with HindIII and BamHI restriction enzymes (based on the map below). On the "gel" below, draw the number of bands, and their respective sizes, that you'd expect for each of the following digestions: uncut, HindIII, BamHI, HindIIIIBamHI double digest (2pts). D) You want to verify that your cloned gene does not have any mutations, so you decide to use Sanger sequencing. How many primers do you need to sequence across the whole gene effectively? Explain your reasoning (1-2 sentences, 2pts).
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Question Text
Describe the gases (including oxygen and carbon dioxide partial pressure percentage ranges), nutrients, and waste products. Explain the relationship between RBC antigens and ABO blood group types. Identify the stages and processes involved in the clotting processes of blood. ESSENTIALS OF ANATOMY AND PHYSIOLOGY (8th edition)
TopicAll topics
SubjectBiology
ClassClass 11