Multiple primer analyzer

For analyzing and comparing multiple primer sequences simultaneously.

Write or paste your primer sequences to the input field (upper window). The analyzer accepts text and table format (can be copied from an Excel file, for example). Note: This analyzer requires at least 2 primer sequences in the input field. For single primers (determination of primer Tm) you can choose the Tm calculator for PCR.

  • A name is required for each primer (eg. Seq1 agtcagtcagtcagtcagtc).
  • The name and sequence string can be separated with either space or tab, as long as the style is the same for all the primers
  • Degenerate primer sequences are also accepted
  • NOTE: If the PCR primer contains desired mismatches, e.g. for creating a mutation or a restriction site, make sure to calculate the Tm only for the correctly matched sequence.

Degenerate primers

A degenerate primer is a mixture of otherwise identical primers where one or more of the positions can have multiple alternative bases. These degenerate bases are represented by specific letters, each denoting one type of variation. For example, a letter B in the primer sequence means that some primers in the mixture can have C in that position, while others can have G or T (see the table below).

The following letters are used to designate degenerate bases in a primer sequence:

B D H K M N R S V W Y
C/G/T A/G/T A/C/T G/T A/C A/C/G/T A/G G/C A/C/G A/T C/T

The results will appear instantly in the output fields (lower windows), and update automatically if you make changes to the sequences. The analyzer will give the following results:

  • Tm (°C)*
  • CG content (%)
  • Length of the primers (nt)
  • Number of individual bases (A, T, C and G)
  • Extinction coefficient (l/(mol·cm))
  • Molecular weight (g/mol)
  • Amount / OD unit (nmol/OD260)
  • Mass (µg/OD260)
  • Primer-dimer estimation**
Type or paste (Ctrl-V) sequence(s) of the primer(s) here in FASTA or two column format.
Number of primers: 0
Parameters for calculation of primer Tm: Parameters for detection of primer dimers:
Primer concentration:µM Value of the sensitivity for dimmer detection (1-10):
(value 1 is maximum sensitive detection, 3 is optimal)
Salt concentration:mM
Results in tabular data format. To export the results: select all (Ctrl-A), copy (Ctrl-C) and paste (Ctrl-V) to Excel sheet.
Results for primer dimer detection. To export the results: select all (Ctrl-A), copy (Ctrl-C) and paste (Ctrl-V).
*
The calculated Tm for a given primer can vary significantly between different calculation methods. This Tm calculator uses a modified nearest-neighbor method based on the method described by Breslauer et al., Proc.Natl.Acad.Sci 83, 3746-50 (1986).
**
The analyzer reports possible primer-dimers based on the detection parameters given below the sequence input window. The dimer information is intended to be used as a preliminary guide when selecting suitable primer combinations. It is not conclusive data. In the actual amplification reaction the primer-dimer formation can vary depending on the PCR conditions.