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the best conditions for PCR

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Hi all

I'm working on a PKU research , and since 3 weeks until now , I,m trying to find the best conditions that i can do in an ARMS-PCR to detect the P281L mutation,
the primers are
forword primer (common) : Tm :58.1
reverse primer (normal) : Tm :60.0
reverse primer (mutant) : Tm : 58.1

first : the PCR reaction is done as the folowing
95   °C                  5 min
95   °C            0:45 min
55-56 °C gradent         0:45 min   } 35 Cycles
72   °C           1:00 min
72   °C                  5 min
°C                   
for positive , negative and blank sampels
the normal and the mutant bands appeared in all lans (expect the blank lans)
with primer dimer and non-specific binding almost at all temperatures so I expect that I have to reduce the primers concentrations and increase the annealing temperatures and use a mix without enhancer
the results was that the normal bands appeared in all the normal lanes and the mutant bonds did not appear at all
so the reaction is repeated twice with some changing and constant 65 c
in the first reaction I just decrease the number of cycles into 30
in the second one I change the time for all the steps as (5 min, 0:10 min, 0:10 min, 0:30min, 5 min,  )
in both reaction the result was the same the normal bands did not appeare
finally the reaction is done by fixing the number of cycles to 30, and the annealing tempreture to 63 c < and changing the tepreture for the three middle steps ((0:10, 0:10, 0:30) & (0:45,0:45, 1:00))
for the first set the result was perfect but with faint bands (the normal band only in the normal lane and the mutant band only in the mutant lane)
how the make the bands sharp
total 237 hits     Asked by: Anonymouse I want to answer  


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