1. Restriction Enzyme Store in -20 C Digestion

2. Restriction Enzyme Buffer NaCl or KCl, TrisHCl, MgCl 2, DTT Different salt: varied activity Buffer supplied with enzyme for 100 % activity

3. Restriction Enzyme Buffer Universal buffer for double or triple digestion some activity reduced Check manual from company if different buffer to be used

4. Restriction Enzyme Methylation Modification commonly found in DNA of bacteria, eukaryote and their virus m6 A, m5 C, m4 C, hm 5C

5. Restriction Enzyme Temperature In general 37 C 67, 25 or 50 C also found

6. Restriction Enzyme Enzyme inactivation Heating 65 C for 15 min Extraction with phenol/chloroform Addition of EDTA

7. Restriction Enzyme Star activity Cleave nucleic acid nonspecifically Factors:Non optimal pH Substitution of Co 2+, Mn 2+, Zn 2+ for Mg 2+ Increase enzyme concentration Reduce salt concentration High glycerol (prevent freezing) Organic solvent higher than 1 %

8. Electrophoresis Analytical method for purification / isolation separation / fractionation identification Electrical field Simple and Rapid Gel medium / Buffer

9. Electrophoresis Agarose / Polyacrylamide gel Detection by staining Ethidium bromide (UV) Acridine orange (UV: ss-orange / ds-green) Silver staining Brilliant blue or Methylene blue

10. Agaro se % in 1x TBE Linear dsDNA (kb) Acryla mide %T in 1xTBE Linear dsDNA (bp) 0.3 0.6 0.7 0.9 1.2 1.5 2.0 5 - 60 1 - 20 0.8 - 10 0.5 - 7 0.4 - 6 0.2 - 3 0.1 - 2 3.5 5.0 8.0 12.0 15.0 20.0 100 - 1000 75 - 500 50 - 400 35 - 250 20 - 150 5 - 100 Range of Separation

11. Electrophoresis Agarose gel Linear polymer of D- and L-galactose Quality varies from batch to batch Separate few hundreds to about 20 kbp Low resolving power Run in horizontal configuration

12. Electrophoresis Agarose gel LMP agarose to be run at 4 C TAE (high MW) or TBE / TPE (low MW) Gel loading dye bromophenol blue, xylene cyanol FF sucrose, ficoll, glycerol

13. Agarose Gel Electrophoresis

15. Polyacrylamide Gel Electrophoresis For protein analysis For smaller DNA fragments High resolving power Run in vertical configuration

16. Polyacrylamide Gel Electrophoresis Acrylamide and Bis-acrylamide APS and TEMED without oxygen Denaturing PAG with Urea/Formamide for ssDNA

17. PAGE

20. Polymerase Chain Reaction Temperature Time Denaturation Annealing Extension

21. Polymerase Chain Reaction Components Thermostable DNA polymerase Template Primer SubstrateBuffer

22. Polymerase Chain Reaction Template small amount (1 ng – 1 ug) free from contamination Primer specific to target / dimer nt length (Tm) / 1-2 primer (s) / GC content modification: mutation / RE site 0.2-1 uM

23. Polymerase Chain Reaction dNTPs 50-200 uM each recommended MgCl 2 1.5 mM recommended effect on primer annealing / Pol activity Gelatin /BSA: enzyme stability DMSO: better denaturation of long target

24. Polymerase Chain Reaction

27. Thermal Cycler

28. Polymerase Chain Reaction Hot start PCR enzyme activity blocked during reaction setup enhance specificity / sensitivity increase target yield by wax, chemical or enzyme Ab

29. Polymerase Chain Reaction Nested PCR Product of previous reaction used as template for next reaction New sets of primers correspond to sequences internal to the previous set

30. Polymerase Chain Reaction Multiplex PCR combined primer sets amplify more than 1 target in 1 tube

31. Polymerase Chain Reaction Real time PCR quantify amplified products comparative assay of initial templates monitor increased fluorescence signal during cycles (not end point)

32. Polymerase Chain Reaction Touch-down / Step-down PCR annealing temp progressively reduced from above Tm to below Tm specific target amplified at high stringency

33. Polymerase Chain Reaction Degenerate PCR sequence of target not known exactly eg. to find new gene or gene family back translate from protein mixed primers with wobbles

34. Polymerase Chain Reaction Degenerate PCR Trp Asp Thr Ala Gly Gln 5' TGG GAY ACN GCN GGN CAR 3' Y = C or T R = G or A N = G, A, T or C Substitute Inosine for N

35. Polymerase Chain Reaction Asymmetric PCR different molar ratio of primers for ssDNA amplification RT PCR Colony PCR In situ PCR