Table 7. General guidelines for standard PCR primers
Length:
1830 nucleotides
G/C content:
4060%
Tm:
Simplified formula for estimating melting temperature (T
m):
T
m = 2C x (A+T) + 4C x (G+C) Whenever possible,
design primer pairs with similar Tm values. Optimal annealing
temperatures may be above or below the estimated T
m.
As a starting point, use an annealing temperature 5C below
T
m.
Sequence:
Avoid complementarity of two or three bases at the
3' ends of primer pairs to reduce primerdimer formation.
Avoid mismatches between the 3' end of the primer and
the target-template sequence.
Avoid runs of 3 or more G or C at the 3' end.
Avoid a 3'-end T. Primers with a T at the 3' end have
a greater tolerance of mismatch.
Avoid complementary sequences within a primer sequence
and between the primer pair.
Commercially available computer software (e.g., Primer
Designer 1.0, Scientific Software, 1990; Oligo, Rychlik and
Rhoads, 1989) can be used for primer design.
Concentration:
Spectrophotometric conversion for primers: 1 A260 unit
= 2030 g/ml
Molar conversions:
'"/>Source:
Page: All 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Related biology technology :1.
HotStarTaq DNA Polymerase2.
QIAGEN Multiplex PCR Handbook3.
TransMessenger Transfection Reagent Handbook4.
Transfection Reagent Selector Kit Handbook