Tools

1. Tools • Enzymes • Vectors • Host • DNA to be cloned

2. Enzymes • Nucleases • Polymerases • Ligases, • Modifying enzymes • Topoisomerase,

3. Exonucleases • Unit Enzyme 1ug DNA at 37C in 60 min in 50 ul reaction* ExonucleaseIssDNA 3-5 ExonucleaseIIIdsDNA 3-5 ExonucleaseVIIssDNA3-5 and 5-3 https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions

4. How they cut Exo III

5. Endonucleases • Dnase I ssDNA, dsDNA • DNA template degradation in transcription reactions • Removal of genomic DNA from RNA samples • DNase I footprinting • Nick Translation • Mung bean nucleases ssDNA • Removal of single-stranded extensions (3' and 5') to leave ligat able blunt ends • Transcriptional mapping • Cleavage of hairpin loops

12. A Paternity Test

13. Basic Structure of DNA to remember

14. Isoschizomer Sph I (CGTAC^G) and Bbu I (CGTAC^G) Neo schizomer Sma I (CCC/GGG) and Xma I (C/CCGGG) Isocaudomer NheIG*CTAG C and AvrIIC*CTAG G C GATC*G G GATC*C

15. Neo schizomer

16. Double digestion Double digestion is a process in which we use two restriction enzymes to cut so that molecules do not snap back on itself or for orientation certainty

18. RIBONUCLEASES RNase A Bovine pancreatic RNase A, Ranapipiens RNaseH RNase H family can be found in nearly all organisms, from archaea to bacteria and eukaryota. Rnase III Double stranded RNA degradation RNAi, microRNA

19. Rnase based therapeutic for cancer Onconase down regulates microRNA expression through targeting microRNA precursors Cell Research (2012) 22:1199–1202. doi:10.1038/cr.2012.67; published online 24 April 2012

20. Rnase H of HIV and HBV as an example 1- Structural Basis for the Inhibition of RNase H Activity of HIV-1 Reverse Transcriptase by RNase H Active Site-Directed Inhibitors. 2010 Journal of Virology

21. Ligase

22. Ligases and Ligastion T4 DNA ligase

23. What Ligase needs to make an efficient ligation • 5 Phosphate is absolute requirement • If removed ligation can not take place • Adjusting the vector insert ratio 1:3 formula? • Contamination in DNA also influence ligation • efficiency

24. Need of Alkaline phosphatase (CIAP) • An enzyme that removes phosphatase from it substrate • Why we need to remove the phosphate if it absolutely required • How DNA is ligated after use of CIAP

25. Need of Alkaline phosphatase (CIAP) • An enzyme that removes phosphatase from it substrate • Why we need to remove the phosphate if it absolutely required • How DNA is ligated after use of CIAP

26. E.coli DNA polymerase I Nick translation

27. Polymerase

28. Klenow fragment Fill in reaction

29. BacteriophageT4 Polymerase Active single-stranded 3'->5' exonuclease (ss DNA) (stronger than that of the Klenow fragment) Fill in Trimming back

30. The T7 polymerase • Enzyme has very high proof reading and polymerization • The enzyme chemically or genetically modified • High processivity, and fast polymerase rate • Used in DNA sequencing

31. Taq DNA polymerase Thermusaquaticus PCR optimization, Pfu DNA polymerase Pyrococcusfuriosus PCR (if DNA has to use in cloning)

32. Terminal De-oxynuclotidylTransferase Probe preparation tailing method

34. AMV reverse transcriptase • HIV-1 reverse transcriptase from human immunodeficiency virus • M-MLV reverse transcriptase from theMoloney murine leukemia virus • AMV reverse transcriptase from the avian myeloblastosis virus

35. RNA polymerases S6 RNA Polymerases T7RNA Polymerases

36. Plasmid Isolation

37. Alkaline de naturation and plasmid isolation

38. Uses of different enzymes • Primer removal from PCR mixtures: Exo1 thermo • prior to PCR product sequencing (see Reference 2) • for one-tube "megaprimer" PCR mutagenesis (see​ Reference 3) • Removal of single-stranded DNA containing a 3'-hydroxyl terminus from nucleic acid mixtures • Assay for the presence of single-stranded DNA with a 3'-hydroxyl terminus (see​ Reference 4) http://www.thermoscientificbio.com/dna-and-rna-modifying-enzymes/exonuclease-i/