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Inclusion of restriction sites in PCR primers

Kevin Mulcahy K.Mulcahy at sheffield.ac.uk
Thu Oct 31 06:17:32 EST 1996

I am trying to clone a cDNA using RT-PCR and have designed a primer pair 
through the help of a computer programme (MacVector). This programme 
apparently (so long as the settings are approriate) designs primer pairs 
which do not form primer-dimers or other primer-primer/primer-product 
artifacts which may inhibit successful PCR amplification. It also 
suggests an optimal annealing temperature to use for the PCR as a 
starting point when using the primers for the first time.

However, I added a further 10 bp onto the 5' ends of each primer so that 
they contained a restriction enzyme site (a different site for each 
primer) to be used for digestion of the amplified product and cloning 
into an appropriate vector for sequencing. I attempted to perform the 
RT-PCR with these modified primers using a range of annealing 
temperatures, annealing times and extension times but I could never 
amplify the desired product.

Therefore, does anyone know what rules should be followed in the design 
of primers when adding restriction sites? Also, are there any general 
rules or suggestions for determining the cycling parameters for PCR when 
using these modified primers?

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