( 6. Mg 2 + concentrat...)HotStarTaq PCR Handbook

6. Mg²+ concentration not optimal Perform PCR with different final concentrationsof Mg²+ from 1.55.0 mM (in 0.5 mM steps) using a 25-mM MgCl2 solution (see Table 2). 7. Enzyme concentration too low When using HotStarTaq DNA Polymerase,use 2.5 units per 100 l reaction. If necessary, increase the amount of HotStarTaq DNA Polymerase (in 0.5-unit steps). When using HotStarTaq Master Mix, use 25 l Master Mix per 50 l reaction. 8. Insufficient number of cycles Increase the number of cycles in steps of 5 cycles (see appendix). 9. Incorrect annealing temperature
or time Decrease annealing temperature in 2C steps. Annealing time should be between 30 and 60 seconds. Difficulties in determining the optimal annealing temperature can be overcome in many cases by performing
touchdown PCR (see appendix). 10. Incorrect denaturation temperature
or time Denaturation should be at 94C for 30 to 60 seconds. Ensure that the initial 15-minute 95C incubation step was performed as described in step 6 of the PCR protocols
. 11. Extension time too short Increase the extension ti
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