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BIO 208 NUCLEIC ACID METHODS

BIO 208 NUCLEIC ACID METHODS. Stephanie Schumaker. Objective. amy E gene. Part of the chromosome in many strains of Bacillus Circular with a size of about 4000 kilobases Gene is about 1.6 kilobases. Procedure. Get the DNA PCR and produce biotin-labeled probe Southern Blot

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BIO 208 NUCLEIC ACID METHODS

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  1. BIO 208NUCLEIC ACID METHODS Stephanie Schumaker

  2. Objective

  3. amyE gene • Part of the chromosome in many strains of Bacillus • Circular with a size of about 4000 kilobases • Gene is about 1.6 kilobases

  4. Procedure • Get the DNA • PCR and produce biotin-labeled probe • Southern Blot • Cloning the amyE gene • Verify and Map • Check Enzyme Activity of Clones

  5. DNA isolation • Centrifugation • Vortex • Glass beads • Reagents • Ethanol • Phenol:chloroform: isoamyl alcohol • Sodium acetate • TE

  6. Quantitation of DNA • DNA A260 nm • Proteins A280 nm • 1.8 to 2.0 ratio is pure DNA

  7. CONCENTRATION • 260nm x 50ug/ml x DF .054 x 50ug/ml x 100 = 270ug/ml 270ug/ml / 1000ul = .27ug/ul About every ul has ¼ ug 4ul = 1ug .7ug/ul and assumed it to be .5ug/ul due to RNA About every ul has ½ ug 2ul = 1ug A reading of 1 @ 260nm = 50ug/ml of double stranded DNA

  8. Restriction Enzyme Cleavage • Enzymatic digestion of the DNA cuts it into fragments of interest • 3 types of enzymes were used • EcoRI • Hind III • Cla I

  9. Gels 1 and 3 • Empty • Marker • Sample A 10-1 • Sample B 10-1 • Sample AW 10-1 • Sample A 10-2 • Sample B 10-2 • Sample AW 10-2 • Empty • Empty • Uncut DNA • Hind III • Marker • EcoR I • Cla I • Empty

  10. Gel 2 Inconclusive

  11. PCR • Denature DNA • Annealing of primers • Synthesis

  12. PCR Gels 1 and 2 • Empty • - DNA • 15ul control • 5ul control • Experimental • Marker • Empty • Empty • Empty • Empty • Empty • Marker • Empty • Experimental • Empty • Empty

  13. PCR and Biotin labeled probe • Nonradioactive • Chemically bonds to base pair of a nucleotide • Taq will incorporate molecules of BIO-deoxycytidine during PCR

  14. Southern Blot • Restriction enzyme cleavage • Denaturation and transfer of DNA to a membrane • Southern Hybridization and detection

  15. Detection Results were inconclusive

  16. Cloning the amyE gene in 5 easy steps 1) Enzyme restriction digest of DNA sample2) Enzyme restriction digest of DNA plasmid vector3) Ligation of DNA sample products and plasmid vector4) Transformation of E. coli with the ligation products 5) Growth on agar plates with selection for antibiotic resistance

  17. Steps 1-3

  18. Plasmid pRL498

  19. Verify and Map • PCR is used to verify positive clones that yield the 433bp product • Make sure the correct fragment was inserted into the vector using enzyme cleavage • Map the 6.3kb amyE plasmid DNA by cleavage and gel electrophoresis

  20. Check for enzyme activity • quantitative alpha amylase assay • break down starch into maltose • use maltose standard curve to determine amount produced

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