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DNA Laboratory Building a DNA Model DNA Fingerprinting DNA Fingerprinting • Detects differences between individuals at the DNA level • Uses restriction enzymes that cut DNA at specific sequences • Differences in position of restriction enzyme cuts yield different DNA fragment sizes • RFLP = restriction fragment length polymorphism Loss of an EcoRI Recognition Site Produces Different Numbers of DNA Fragments Gel Electrophoresis Direction of Migration Larger fragments move more slowly; smaller fragments move more rapidly Submerging the gel Pouring the agarose Loading the gel Using the Micropipettor Set volume to 35 ul Attach a clean tip Push plunger to first stop Insert tip into liquid Release plunger SLOWLY Position over well in gel Push plunger SLOWLY to second stop to fill well Discard tip in orange bag Model will show Replication, Transcription and Translation replication (S phase) DNA transcription (G1 and G2 phases) RNA translation Protein Completed Model • One DNA double helix, two chains of 9 nucleotides each – one chain can have any sequence – second chain must be complementary • One chain of mRNA, 9 nucleotides long – must be complementary to one of the two DNA chains (DNA template is 3’5’, mRNA is 5’3’) • Three tRNAs with appropriate amino acids attached – tRNA anticodons must match mRNA codons Structure of a Nucleotide Components Phosphate Sugar Base Phosphate at 5’ position 3’ position Link the phosphate of one nucleotide to the sugar of the next nucleotide to produce a DNA chain. 5’ Replication Pair A with T and G with C to produce the second chain. One chain is 5’3’ Other chain is 3’5’ 3’ 3’ 5’ 5’ DNA polymerase adds nucleotides to the growing chain in a 5’3’ direction. 3’ 3’ 5’ 5’ 3’ 3’ 5’ Your completed model will have two chains, each with 9 nucleotides. 5’ Transcription One DNA chain is used as a template. Template is read 3’5’ RNA is produced 5’3’ 3’ 3’ Base Base in DNA in RNA A U T A G C C G 5’ 5’ RNA polymerase adds nucleotides to the growing chain in a 5’3’ direction. 3’ 3’ 5’ 5’ 3’ 3’ 5’ Your completed messenger RNA will have 9 nucleotides. Three Types of RNA rRNA DNA transcription mRNA tRNA Joins with proteins Specifies order of Carries amino to form ribosome amino acids acids Structure of transfer RNA amino acid attachment site mRNA anticodon Anticodon on tRNA recognizes codon on mRNA to bring amino acid into place. Each group of 3 nucleotides on mRNA is a codon. Codons are read from 5’3’. tRNA anticodons, from 3’5’ match the codons. codon anticodon 3’ 5’ 3’ 5’ Read the CODON from 5’3’ to determine the amino acid carried by the tRNA. Serine Serine Your completed model will have 3 tRNAs, one for each codon in mRNA. Show the name of the amino acid carried by each tRNA. CLEAN UP! • After pouring gel, fill flask with water and return to bath • Put used tips in the orange bags • Rinse and dry the gel tray, comb and rubber dams • Leave buffer in electrophoresis chamber • After analysis, discard staining sheets and agarose gels into trash • Put all paper clippings into trash • Make sure the sinks stay clean Worksheet • Attach your completed DNA model inside a legal-sized folder • Complete pages 91-92 from lab manual • Be sure your instructor checks both of these items before you leave the lab