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Sequenziamento: metodo di Sanger con ddNTP

Sequenziamento: metodo di Sanger con ddNTP. Sequenziamento con dideossonucleotidi. Sequenziamento automatizzato del DNA. Clonaggio:. Inserto. Vettore. +. Ligazione. GAATTC CTTAAG. GAATTC CTTAAG. G p AATTC CTTAA G. p AATTC G. AATTC G. G AATTC CTTAA p G. G CTTAA p.

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Sequenziamento: metodo di Sanger con ddNTP

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  1. Sequenziamento: metodo di Sanger con ddNTP

  2. Sequenziamento con dideossonucleotidi

  3. Sequenziamento automatizzato del DNA

  4. Clonaggio: Inserto Vettore + Ligazione

  5. GAATTC CTTAAG GAATTC CTTAAG GpAATTC CTTAA G pAATTC G AATTC G G AATTC CTTAApG G CTTAAp G CTTAA DNA ligasi pAATTC G G CTTAAp EcoRI Trattamento del vettore con fosfatasi fosfatasi

  6. Vettori plasmidici (pBR322)

  7. Vettori plasmidici (pUC18)

  8. Colonie Blu Colonie bianche β-galattosidasi ATTIVA • Ceppo di E.coli Lac Z mutante • Vettore pUC LacZ parziale Complementano • Ceppo di E.coli Lac Z mutante • Vettore LacZ interrotto dal clonaggio Non c’è complementazione β-galattosidasi INATTIVA β-galattosidasi ATTIVA Metabolizzato Terreno + X-Gal Non metabolizzato β-galattosidasi INATTIVA X-Gal: 5-Bromo-4-Cloroindolil-β-galattoside (incolore)

  9. 1° ciclo di PCR DNA genomico 5’ 3’ 3’ 5’ Primer reverse Primer forward 5’ 3’ 3’ 5’ DNA genomico

  10. ETEROGENEITA’ DEL TEMPLATO : ETEROZIGOSI PER UNA DELEZIONE

  11. F P M C/C G/- C/- LOSS OF HETEROZYGOSITY

  12. RNA sequencing Dopo trascrizione inversa

  13. 6 8 7/- 7/- -/- 6 7 8 CTT

  14. Figure 1: Illumina Flow Cell Figure 2: Prepare Genomic DNA Sample DNA Adapters Figure 3: Attach DNA to Surface Figure 4: Bridge Amplification Adapters DNA Dense lawn of primers

  15. Figure 5: Fragments Become Double Stranded Figure 6: Denature the Double-Standed Molecules Attached terminus Attached Attached terminus Free terminus Attached Figure 7: Complete Amplification Figure 8: Determine First Base Clusters Laser

  16. Figure 9: Image First Base Figure 10: Determine Second Base Figure 11: Image Second Chemistry Cycle Figure 12: Sequencing Over Multiple Chemistry Cycles Figure 13: Align Data Laser GCTGA...

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