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Molecular Week 10 Notes

by: Kiara Lynch

Molecular Week 10 Notes Bio 413

Marketplace > La Salle University > Biology > Bio 413 > Molecular Week 10 Notes
Kiara Lynch
La Salle

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About this Document

Week 10 Notes
Dr. Stefan Samulewicz
Class Notes
25 ?




Popular in Molecular

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This 2 page Class Notes was uploaded by Kiara Lynch on Tuesday April 5, 2016. The Class Notes belongs to Bio 413 at La Salle University taught by Dr. Stefan Samulewicz in Spring 2016. Since its upload, it has received 14 views. For similar materials see Molecular in Biology at La Salle University.


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Date Created: 04/05/16
Molecular Week 10 Notes  Process of isolating colonies- Plating o Spread out colonies of bacteria containing recombinant plasmids o Nitrocellulase paper on top- treated paper o O/N incubation o Remove paper- some bacteria will stick o Lyse bacteria, denature DNA o Plaque formation- each plaque is a colony of 1 clone o Incubate with probe and wash  Radioactively labeled DNA probe  Labeled nucleotide- attach to complementary sequence  Or antibody- bind to protein  Gives colonies containing plasmid of interest o Expose paper to photographic film o Colonies with at least part of genome o Expression vector- produces protein associated with the protein of interest o Libraries contain many clones in vials in a freezer; plate clones to access them  Dideoxy chain termination sequencing technique - find overall sequence of gene o Codons for amino acids o Look for regions that show a low level of degeneracy o Synthesize all 16 possibilities labeled probe  screen library and isolate the complete clone and use it to get complete, correct nucleotide sequence o Dideoxynucleotide  Missing 3’ OH  New nucleotide can no longer be added- chain termination o Melt  single strand  add primer  allow polymerase to start synthesizing new strand  randomly introduce different dideoxynucleotides  chain termination  different length sequences  run 4 tubes of DNA on gel  separate by size  radioactive C’s are most active o Acrylamide with radioactive samples  Bands separated by 1 nucleotide in length o Label each dideoxynucleotide individually  Different color fluorescent tag  seen differently by spec  1 tube and run in 1 lane  Spec at bottom of gel  light up bands as they pass by  able to detect which nucleotide passed by  Steps to sequence a genome o Extract DNA o Restriction enzyme to cut  make library o Pick clones o Grow o Sequence o Repeat with different restriction enzyme to find overlap  put in order  Genomic library o Only about 1.5% of human genome is coding info o 3 stop codons out of 64 codons  1 is randomly introduced about every 20 codons o Open reading frame- no stop codon  sequence codes for protein  PCR- polymerase chain reaction o Amplify particular segments of DNA o 1. Melting o 2. Annealing – bind to sequence and create free 3’ end o 3. Elongation – Taq polymerase adds nucleotides in correct order creating second sequence o Primers  Clone directly- TA cloning  Anneal to plasmid o 1 cycle  Double stranded chromosomal DNA  separate DNA strands and add primer  Produces two double stranded DNA molecules o 2ndcycle  Separate DNA strands and anneal primer  DNA synthesis rd Produces 4 double stranded DNA molecules o 3 cycle  Produces 8 double stranded DNA molecules


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