Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 2: MATERIALS & METHODS2.3.2.1 General PCR Conditions for Designed Non-Degenerate PrimersTable 22: PCR reaction components for amplification with the designedprimers shown in table 20.ReactionConcentrationComponents5X buffer 5X 5Water - 16Primer forward 400 ng 1Primer reverse 400 ng 1Taq Polymerase 2 units 1DNA 50 – 150 ng 1Total 25Components of atypical 25 μlreaction (μl)Table 23: PCR cycling profile for amplification with the designed primers shown intable 20.PCR Step Temperature ( o C) Time (mins) CyclesInitial94 8 1Denaturation*Denaturation 94 1Annealing 65 1 30Extention 72 2Final Extention 65 172 10 1Soak^ 25 - -* A hot start was performed at beginning of PCR cycling, whereby Taq polymerasewas added after temperature of PCR machine reached greater than 90 o C.^ The PCR program ends at the Soak step where the PCR reaction stops and isincubated at 25 o C until ready for gel electrophoresis2.3.2.2 Degenerate PCR Conditions for Type I PKSSeparate reaction and amplification conditions for degenerate PCR were applied toamplify the 0.75 kb product representing the ketosynthase (KS) gene as specified inthe original report (Liu & Shen, 2000). Only in the presence of 20 % glycerol was aPCR product of the correct size produced as specified in the original publishedmethod (Liu & Shen, 2000). Absence of 20% glycerol yielded no amplificationproduct._____________________________________________________________________58