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?