Lab 3 - Lab 3 Nucleic acid Nucleotide: 5 end 5 3 5 3 H...

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Lab 3
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Nucleic acid 3’ end 5’ end 3’ end 3 H bonds 2 H bonds Phosphodiester bond DNA is double stranded!!! 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ Direction of Synthesis 5’ 3’ Nucleotide: 5’ end Be familiar with the basic structure of DNA!
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Cloning You want to amplify a molecule You often work with mixed and huge population of molecules and cells Source DNA Plasmid DNA LacZ gene Encodes β - Galactosidase (blue product) Multi cloning site contains unique restriction sites Antibiotic resistance gene (amp R ) Origin of replication (induces replication in E. coli ) Plasmid (“shuttle molecule”)
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Restriction enzymes recognize specific DNA sequences Sites are often palindromes: same sequence on both strands Cleave phosphodiester bonds (most) generate sticky ends (overhangs) Cut source and plasmid with the same enzyme Restriction Circular plasmid Source DNA Hind- III Hind- III Small pieces of Source DNA Linear plasmid
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Annealing and Ligation Linear plasmid Source DNA + 1. Annealing ( hydrogen bonds between bases of neighboring strands = base pairing) 2. Ligation (ligase forms phosphodiester bonds between nucleotides on the same strand Ligation products include: religated plasmid, plasmid with insert X plasmid with insert Y, plasmid with insert X + Y, . ... = recombinant molecules
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Transformation (here: ligation reaction + Escherichia coli ) Recombinant DNA plasmids Contain insert(s) religated plasmid NO insert + (competent) E. coli 1. Mix ligation reaction with competent E. coli . 2. Incubate for 30 min on ice 3. Heat shock for 2 min @ 42 o C 4. Incubate for >15 min @ 37 o C in pre-warmed LB 5. Plate transformation reaction on LB ± ampicillin and X-gal 6. Incubate over night @ 37 o Cin²LB pBLU No insert: lacZ gene intact β Gal converts X-Gal to blue product = blue colonies pBLU i With insert: lacZ gene disrupted β Gal not functional no blue product = white colonies
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LB lawn > 99.99% Transformation efficiency is LOW!!! < 0.01% LB Amp/X-gal Isolated colonies Use of controls: Bacteria contaminated LB medium contaminated X-gal amp R functional Correct plasmid
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Enzymes are proteins Genomic DNA (nucleus, double-stranded, exons + introns) transcription Pre-RNA (nucleus, single stranded, exons + introns mRNA (nucleus, single stranded, just exons splicing translation 3 nucleotides code for 1 amino acid Proteins cytosol synthesized at ribosomes
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Polar, nonpolar Hydrophobic, hydrophylic Amino acid pKa 5.3 (equilibrium at pH of 5.3) Example: pH 4 means more free H+ in solution so the equilibrium is shifted to the left Positively charged R groups (Arginine, Lysine): (NH 3 + NH 2 + H + ) pK a ~10
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Protein conformation H bonds H bonds S=S bonds H bonds S=S bonds Peptide bonds DNA Pre RNA mRNA Side chain interactions polypeptide interactions sequence Back bone interactions Enzymes need to bind a substrate (3D structure matters, key + lock) Amylase + Starch ES Amylase + Maltose E + S ES E + P Enzyme substrate complex depends on 3D structure
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Lab 3 - Lab 3 Nucleic acid Nucleotide: 5 end 5 3 5 3 H...

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