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Keck
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Template/Primer Guidelines
Primer Selection
Guidelines
The following is a list of
parameters to take into account when designing primers for use under our
automated sequencing conditions:
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20-30 nucleotides in length.
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50% G/C content.
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G & C "clamps" on the 5' and 3'
ends (at least a single G or C residue.)
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Avoid multiple Thymidine
residues on 3' and 5' ends.
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Avoid primers with long runs
(more than 4) of a single base.
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Avoid primers with secondary
structures or that can hybridize to form dimers or hairpins. These can
be easily predicted if a primer design program is used such as primer select.
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Melting temperature 55-65o
C.
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Check primers for specificity in
annealing to template.
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Primers should be located at
least 30-40 bases upstream of the area of interest in the sequence read
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