210 likes | 318 Views
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
E N D
BIO 208NUCLEIC ACID METHODS Stephanie Schumaker
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
Procedure • Get the DNA • PCR and produce biotin-labeled probe • Southern Blot • Cloning the amyE gene • Verify and Map • Check Enzyme Activity of Clones
DNA isolation • Centrifugation • Vortex • Glass beads • Reagents • Ethanol • Phenol:chloroform: isoamyl alcohol • Sodium acetate • TE
Quantitation of DNA • DNA A260 nm • Proteins A280 nm • 1.8 to 2.0 ratio is pure DNA
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
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
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
Gel 2 Inconclusive
PCR • Denature DNA • Annealing of primers • Synthesis
PCR Gels 1 and 2 • Empty • - DNA • 15ul control • 5ul control • Experimental • Marker • Empty • Empty • Empty • Empty • Empty • Marker • Empty • Experimental • Empty • Empty
PCR and Biotin labeled probe • Nonradioactive • Chemically bonds to base pair of a nucleotide • Taq will incorporate molecules of BIO-deoxycytidine during PCR
Southern Blot • Restriction enzyme cleavage • Denaturation and transfer of DNA to a membrane • Southern Hybridization and detection
Detection Results were inconclusive
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
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
Check for enzyme activity • quantitative alpha amylase assay • break down starch into maltose • use maltose standard curve to determine amount produced