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Sanger Sequencing

Sanger Sequencing. 1) Get Some DNA To Sequence . *le pick…. *le smash…. *le DNA in le solution. *le precipitate…. 2) Add a Primer That Binds To Area You Want to Sequence . * l e primer. *le DNA in le solution. Base pair with gene to sequence. 3’ end for adding nucleotides.

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Sanger Sequencing

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  1. Sanger Sequencing

  2. 1) Get Some DNA To Sequence *le pick… *le smash… *le DNA in le solution *le precipitate…

  3. 2) Add a Primer That Binds To Area You Want to Sequence * le primer *le DNA in le solution Base pair with gene to sequence 3’ end for adding nucleotides

  4. 3) Make Many Copies of the Gene *le DNA Polymerase *le free nucleotides (dNTPS) *le DNA+ le Primers Heating Cycle: • 94oC to break H-bonds • 65oC Primers anneal to ssDNA • 72oC DNA Polymerase copies genes using dNTPS • Repeat Many Times

  5. How Heating Cycle Works Heating Cycle: • 94oC to break H-bonds • 65oC Primers anneal to ssDNA • 72oC DNA Polymerase copies genes using dNTPS • Repeat Many Times *le DNA *le H-bonds

  6. How Heating Cycle Works Heating Cycle: • 94oC to break H-bonds • 65oC Primers anneal to ssDNA • 72oC DNA Polymerase copies genes using dNTPS • Repeat Many Times

  7. 4) Copy Gene Using Doubly Deoxidised, Florescent NTPs *le DNA Polymerase *le free nucleotides (dNTPS) *le many copies of gene + primers Fluorescent dye (A=Red, C= Blue…) Doubly deoxidised (no 3’ oxygen to form next bond)

  8. How ddNTPs Work Heating Cycle: • 94oC to break H-bonds • 65oC Primers anneal to ssDNA • 72oC DNA Polymerase copies genes using dNTPS • Repeat Many Times *le DNA *le H-bonds

  9. How Heating Cycle Works Heating Cycle: • 94oC to break H-bonds • 65oC Primers anneal to ssDNA • 72oC DNA Polymerase copies genes using dNTPS • Repeat Many Times

  10. 5) Repeat Many Times To Get Copies of Different Lengths all Ending in a ddNTP *le messy mess

  11. 6) Sort DNA Segments By Size With Gel Electrophoresis *le not messy mess *leagarose gel *leshockieshockie

  12. 7) Use A Laser To “Read” Sections and Sequence DNA *le sequence *le cool laser

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