1. BIO 208 NUCLEIC ACID METHODS Stephanie Schumaker

2. Objective Alpha Amylase Bacillus licheniformis amyE gene Escherichia coli

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 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

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 –EcoR I –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  Uncut DNA  Hind III  Marker  EcoR I  Cla I  Empty

10. Gel 2 Inconclusi ve

11. PCR Denature DNA Annealing of primers Synthesis

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

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 amy E 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