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Digesting DNA with restriction endonuclease

Digesting DNA with restriction endonuclease Mix the DNA solution and reaction buffer (Total volume: Y ml) DNA solution X m l ( 5 m l) 10x reaction buffer 1/Y m l ( 1 m l) Restriction Enzyme 1 m l ( 1 m l) Add ddH 2 O to Y m l ( 3 m l)

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Digesting DNA with restriction endonuclease

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  1. Digesting DNA with restriction endonuclease • Mix the DNA solution and reaction buffer (Total volume: Y ml) • DNA solution X ml ( 5 ml) • 10x reaction buffer 1/Y ml ( 1 ml) • Restriction Enzyme 1 ml ( 1 ml) • Add ddH2O to Y ml ( 3 ml) • Incubate at 37℃ for 1 hour (depend on restriction enzymes) • Argarose gel electrophoresis

  2. Recombinant DNA Technology • Gene cloning or Molecular cloning • The recombinant DNA construction • Transformation • Identification

  3. The recombinantDNA construction Transformation Identification

  4. Restriction Endonucleases

  5. Restriction Endonucleases

  6. Restriction Endonucleases

  7. Restriction Endonucleases

  8. Restriction Endonucleases • Nomenclature • Eco RI EscherichiacoliR • Hpa I Haemopophilus parainfluenzae • Palindromic sequence • Types of cut ends • 5’ phosphate extension • 3’ hydroxyl extension • Blunt end

  9. Fig 4.2 Palindromic sequence

  10. Fig 4.3

  11. Table 4.1

  12. Isochizomer • Different restriction enzymes recognize different sequences • Cleave within the same target sequences • Sau 3A, Mbo I & Bam HI (5’…GATC…3’) • SalI & XhoI (5’-GTCGAC- & 5’-CTCGAG-)

  13. Fig 4.4

  14. Fig 4.5

  15. Fig 4.6

  16. Ligases • E coli • T4

  17. Fig 4.7

  18. Table 4.3

  19. Klenow fragment

  20. 3’ to pyrimidine 3’ to guanine

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