Restriction Enzyme Store in -20 C Digestion

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

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

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

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

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

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 %

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

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

Agaro se % in 1x TBE Linear dsDNA (kb) Acryla mide %T in 1xTBE Linear dsDNA (bp) Range of Separation

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

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

Agarose Gel Electrophoresis

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

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

PAGE

Polymerase Chain Reaction Temperature Time Denaturation Annealing Extension

Polymerase Chain Reaction Components Thermostable DNA polymerase Template Primer SubstrateBuffer

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 uM

Polymerase Chain Reaction dNTPs uM each recommended MgCl mM recommended effect on primer annealing / Pol activity Gelatin /BSA: enzyme stability DMSO: better denaturation of long target

Polymerase Chain Reaction

Thermal Cycler

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

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

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

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

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

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

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

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