Length

We recommend that you keep your primer length between 17 and 25 nucleotide-long.

Sequence specificity

• Make sure that there is only one binding site in the genome for your primer.
• Be sure to choose a primer whose sequence is in your vector.
• We do not recommend to use degenerate primers.
• The primer should match the template exactly. Near the 3' end an exact match is essential, especially the last 8 bases.

If you design a primer from the output of a larger sequence, remember that the first 50-500 bases are more likely to be accurate, and sequence beyond 500 bases is more likely to have errors. Therefore, unless you have sequence information from the opposite strand or overlapping data from another sequence, be conservative and choose your primer in the safer region, preceding base 500.

Estimated melting temperature (Tm)

Primers for cycle sequencing should have a Tm of 50-70°C, ideally between 55-65°C.

Please be aware that we add betaine to each reaction, which may lower both the Tm and annealing temperature of your primer.

Our thermocycling protocol anneals at 50°C and extends at 60°C. If the Tm of your primer is on the low side, please consider redesigning a longer primer. When the Tm is too low, the primer may anneal incorrectly or not at all. A high Tm can be OK if there are not long strings (>3) of Gs or Cs that can bind quickly, often incorrectly, and very tightly. Your G+C content should be approximately 50%.

Be aware that primer design software packages calculate Tms based on some theoretical model that does not always yield actual experimental Tms. Base stacking and nearest neighbor models give the most accurate theoretical Tms. However, we have found that two fairly simple equations can give useful results.

The McConaughy equation (Biochemistry 8: 3289-3295, 1969), modified for cycle sequencing:

Tm = 60 + 41(G + C)/L - 500/L where L = length of primer

The Wallace equation (Nucleic Acids Research 6: 3543-3557, 1979):

Td = 2(A + T) + 4(G + C) Note: This is actually dissociation temperature.

Remember that all calculated Tms are only estimates. They are meant only as starting points and do not guarantee success. We recommend that you avoid the extremes and choose a Tm between 55-65°C, if possible.

The Tm of the 5' end should be similar to the Tm of the 3' end.

A quick way to determine the Tm at each end of the primer is to count the number of A/T bases and C/G bases within 6 nucleotides of each end. Choose the primer with the most similar numbers. This will help ensure that the primer anneals flat with the template strand.

Primer sequence

Avoid primers that can form hairpin loops or primer-dimers. Also avoid stretches of more than 2 identical bases (especially C or G), particularly at the 3' end. This can cause slippage or mismatch during annealing, resulting in a bulge in the primer/template hybrid which could prevent the polymerase from priming.

Plasmid-universal primer compatibility

Please be sure your plasmid samples are compatible with our universal primers before submitting samples for sequencing with our primers.