BIO 208 NUCLEIC ACID METHODS Stephanie Schumaker

Objective Alpha Amylase Bacillus licheniformis amyE gene Escherichia coli

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 GGet the DNA PPCR and produce biotin-labeled probe SSouthern Blot CCloning the amyE gene VVerify and Map C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 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 –EcoR I –Hind III –Cla I

Gels 1 and 3  Empty  Marker  Sample A  Sample B  Sample AW  Sample A  Sample B  Sample AW  Empty  Uncut DNA  Hind III  Marker  EcoR I  Cla I  Empty

Gel 2 Inconclusi ve

PCR Denature DNA Annealing of primers Synthesis

PCR Gels 1 and 2  Empty  - DNA  15ul control  5ul control  Experimental  Marker  Empty  Marker  Empty  Experimental  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 sample 2) Enzyme restriction digest of DNA plasmid vector 3) Ligation of DNA sample products and plasmid vector 4) Transformation of E. coli with the ligation products 5) Growth on agar plates with selection for antibiotic resistance

Steps 1-3

Plasmid pRL498

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 amy E 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