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DIY Molecular Cloning I : DNA Overview, Extracting DNA,

DIY Molecular Cloning I : DNA Overview, Extracting DNA, Finding genetic sequences, Designing a cloning strategy. C. Rouskey Jan.2014. Intros. ...name and background, what you hope to learn... Me GETit. Outline. DNA Structure and Function/DNA Extraction

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DIY Molecular Cloning I : DNA Overview, Extracting DNA,

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  1. DIY Molecular Cloning I: DNA Overview, Extracting DNA, Finding genetic sequences, Designing a cloning strategy C. Rouskey Jan.2014

  2. Intros • ...name and background, what you hope to learn... • Me • GETit

  3. Outline • DNA Structure and Function/DNA Extraction • Sequences on the Web/Determining your GOI • Choosing a plasmid • Components (OriC, Promoter, Reporters, GOI) • Designing a cloning Strategy • Cloning System? Eukaryotic or Prokaryotic • Cloning • GETit Cloning Work

  4. DNA

  5. DNA Basics • Deoxyribonucleic Acid • Stores information • Made up of nucleotides • Adenine (p), Cytosine (py), Thymine (py), Guanine (p) with a deoxyribose backbone; RNA T = U(racil) • These nucleotides bind in complement to each other via hydrogen bonds (G:::C; A::T(U)) • Hydrogen bonds are fairly weak when taken on their own, but the cumulative forces of these bonds make DNA stable • 5' and 3' ends • Two strands running in the opposite direction • Sequences are reverse complement

  6. What kind of DNA? • Prokaryotic or Eukaryotic • Genomic • Plasmid

  7. DNA Replication Where does it occur? How does it occur (overview)? Major enzyme? What are the results?

  8. DNA Transcription Where does it occur? How does it occur? Promoter, ATG What are the results?

  9. Start and Stop Codons

  10. DNA Translation Where does it occur? How does it occur? Ribosomes/tRNA, Start/Stop Codons What are the Results? PROTEINS

  11. Overview: Cloning with REs What is molecular cloning? tools used to create recombinant DNA molecules General steps: Amplify (via PCR) gene with RE sequences present Digest PCR product and plasmid/Purify Ligate PCR product into Plasmid Transform E. coli – look at expression or purify plasmid

  12. Molecular Biologist Toolkit Genetic Databases – resource for sequences Online analysis tools – primer design, restriction assessment DNA/RNA – the genetic starting material Plasmids – functional holding place for gene Primers – directs DNA amplification, targets specific genes Enzymes – used in the amplification cutting and ligation of DNA. Host Strains – amplify and express your GOI

  13. Gene search: http://www.pubmed.com Restriction analysis: http://tools.neb.com/NEBcutter2 Primer assessment: http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/ Composite program: Serial Cloner Online Resources

  14. Plasmids Autonomously reproducing circular DNA Origin of replication determines copy number (OriC) Careful not to create a bio-burden on your cells

  15. Plasmids Promoter - drives the transcription of GOI Multiple cloning Site – Space to pop in GOI downstream of promoter Reporters – let you assess if GOI is being expressed Purification Tags – helpful in downstream purification of proteins (useful tool for functional protein studies

  16. Primers Strands of DNA that serve as the starting point for DNA synthesis

  17. Host Strains E. coli is the basic cloning host for growing plasmid and expressing prokaryotic proteins. E. coli can be used to propagate plasmid containing eukaryotic genes but expression is not really useful (post-translational modifications present in Euks) Competent cells! E. cloni 10G (super easy), Can make your own competent cells After plasmid is made, pop it into E. coli

  18. GETit Specific Cloning (example)

  19. Quick GETit background Bacteriophage therapy Lytic vs. Lysogenic Phage (for more info visit http://getitproject.org/getit-project-meetup-112313/

  20. Cloning (GETit) 1. Find the genetic sequences of interest http://www.pubmed.com a. are there phage in the N. gonorrhoeae genome?

  21. Cloning Strategy Find a plasmid that you can clone into: What are you trying to accomplish? What kind of gene is it? What do you want to do with the genetic material? Order plasmid from addgene (http://addgene.com) We want a moderate copy number, promoter, antibiotic resistance gene

  22. 5'-TCACACTGCACAGCCCCGCTGCCGACATCGAATGCGGCAACGGCGAATACATCCGAATCACGTCCACGCTCGAGCGCGAATAAATGAACAGCACCGTCCCCGCGAACAACAGCCCCCTGCAACACGCACTGGCAAAGCTGACAGAACGCGAAACTGCCGCCGTCTCCCGCCAACTCGACCCCGCCCGATGCGACCCCGGATTTTTACCCTTTCACGCCTTCGCAAGAAGCATCGGCACGGAAGAGGGCTGGGACTTTGCCGAAACCGACGAAGCCCGCCGCAACCTCATCGCAGGCTTTGCCGAAATCCACGCCCGAAAAGGCACGCCGTACGCCATCCGCGCCCTCTTCCCCATCTTGCGGCTGGGCGAAATCCAAATTATCGAACGCGACGGCGAGTTCAAGTGGGACGGCTCGGTCTTGTTCGACGGCAGCCGCACATTCGGCAGGCGCGAGGGTGACTGGGCGGAATACCGCATTGTCTTAACGCGCCCCGTCAGCATCCGCCAAACCGCCCGCATCCGCGCCATGTTGGCGGAAATCGCCCCCTTGCGGTGCGAACTTACCGCGCTCGACTACCGCAACCATCCCCACCGCTGGAACGGCAAAATCCGCTTTAACGGCGAATACGGTTTCGGCACGACATAACGCGCCCCCGAAAATCAACAAACAAAAGGAAGCCCCAAAATGGCAAACGCAACCGAACAAAACCAATTCGACCAAGCCGTCCGCC-3'5'-TCACACTGCACAGCCCCGCTGCCGACATCGAATGCGGCAACGGCGAATACATCCGAATCACGTCCACGCTCGAGCGCGAATAAATGAACAGCACCGTCCCCGCGAACAACAGCCCCCTGCAACACGCACTGGCAAAGCTGACAGAACGCGAAACTGCCGCCGTCTCCCGCCAACTCGACCCCGCCCGATGCGACCCCGGATTTTTACCCTTTCACGCCTTCGCAAGAAGCATCGGCACGGAAGAGGGCTGGGACTTTGCCGAAACCGACGAAGCCCGCCGCAACCTCATCGCAGGCTTTGCCGAAATCCACGCCCGAAAAGGCACGCCGTACGCCATCCGCGCCCTCTTCCCCATCTTGCGGCTGGGCGAAATCCAAATTATCGAACGCGACGGCGAGTTCAAGTGGGACGGCTCGGTCTTGTTCGACGGCAGCCGCACATTCGGCAGGCGCGAGGGTGACTGGGCGGAATACCGCATTGTCTTAACGCGCCCCGTCAGCATCCGCCAAACCGCCCGCATCCGCGCCATGTTGGCGGAAATCGCCCCCTTGCGGTGCGAACTTACCGCGCTCGACTACCGCAACCATCCCCACCGCTGGAACGGCAAAATCCGCTTTAACGGCGAATACGGTTTCGGCACGACATAACGCGCCCCCGAAAATCAACAAACAAAAGGAAGCCCCAAAATGGCAAACGCAACCGAACAAAACCAATTCGACCAAGCCGTCCGCC-3' GOI: phage tail protein I (187aa)

  23. Enzymes DNA Polymerase (used to amplify template) Restriction endonucleases cut at specific sites Ligase

  24. Deciding where to clone? 1. I like BamHI and EcoRV (can I cut them together?) http://66.155.211.155/nebecomm/DoubleDigestCalculator.asp BamHI EcoRV 2. Analyze Gene Sequence - Are there restriction sites internally? http://tools.neb.com/NEBcutter2/!

  25. Primer Design 1. Determine Primer sequence for PCR... 5' end... Sequence: 5' -TCACACTGCACAGCCCCGCTGCCGACATCGAATGCGGCAA Primer: 5' - CCGCTGCCGGATCCGAATG 3' end... Sequence: 5' - AAAACCAATTCGACCAAGCCGTCCGCC – 3' 3' - TTTTGGTTAAGCTGGTTCGGCAGGCGG – 5' Primer: 3' - TTTTGGTTGATATCGTTCGGCAGGCGG – 5' 5' - GGCGGACGGCTTGCTATAGTTGGTTTT – 3'

  26. DNA Sequence 5' – TCACACTGCACAGCCCCGCTGCCGACATCGAATG -3' Primer 5' - CCGCTGCCGGATCCGAATG Complement 3' - AGTGTGA.... Reverse Complement 5' – CATTCGAT....

  27. Now...PCR

  28. Restriction Digest Set up a restriction digest reaction using BamHI and EcoRV Digest Plasmid and PCR product Analyze digest on gel

  29. Ligate and Transform!

  30. 18 hours later. . .

  31. Sequencing (confirm protein) Protein Expression/Analysis via PAGE Protein Purification Downstream Applications

  32. Experiment 1. Find the genetic sequence http://banana-genome.cirad.fr/musa 2. Design primers 3. Extract DNA from Musa acuminata...(Protocol) 4. PCR

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