iOligo EazyPrimer

iOligo EazyPrimer calculates basic PCR primer parameters and identifies secondary structures as you type your oligo’s sequence. The sequence's length cannot exceed 90 mers.

The following parameters are calculated:
Sequence length, CG content, molecular weight, and Tm with and without salt adjustment. Default salt concentration is set to 50 mM Na+.

The following formulas are used for calculating Tm. If sequences less than 14 nucleotides then the same formula is used for standard and salt adjusted Tm:

Tm = 2*(nA+mT)+4*(pG+qC)

For longer sequences the formulas for standard and salt adjusted Tm are:

Tm=64.9+41*(pG+qC-16.4)/(nA+mT+pG+qC)
Tm=100.5+(41*(pG+qC)/(nA+mT+pG+qC))-(820/(nA+mT+pG+qC))+16.6*log([Na+])

Where n,m,p and q represent the number of respective base.

Secondary structures with stem length > 5 will be identified. A structure is considered a palindrome if loop length < 5 otherwise a hairpin structure is indicated.

Standard nomenclature with square brackets, indicating self-annealing areas, is used. For example, if the sequence AAACCCCCAAAAAGGGGG is entered then its secondry structure will be displayed as AAA[CCCCC]AAAAA[GGGGG]. Similarly, the palindrome sequence AAACCCCCGGGGG will be displayed as AAA[CCCCCGGGGG].

Note that EazyPrimer will find any secondary structure, if there is one. However, for sequences containing multiple structures only the first found will be displayed.