SME
Biochemistry · Problem type
Melting temperature of a primer (Wallace rule)
Use this for “A 16-base primer has 7 A or T and 9 G or C. Estimate its melting temperature and GC content.”
Chart
A + T: 22 °C
G + C: 36 °C
This is an example. Type your own values to start a new problem.
Tₘ (°C)
=
2
×
nAT
+
4
×
nGC
n
Primer length in bases
calculated
%GC
GC content
calculated · %
Formulas
n
=
nAT
+
nGC
20 = 11 + 9
%GC
=
100
×
nGC
n
45
=
100
×
9
20
Tₘ = 2 × nAT + 4 × nGC
58 = 2 × 11 + 4 × 9
Assumptions
•
The Wallace rule is a rule of thumb for primers of about 14 to 20 bases in about 50 mM salt.
•
Each A–T pair adds 2 °C and each G–C pair, held by three hydrogen bonds, adds 4 °C.
•
Longer primers, other salt levels or mismatches need the nearest-neighbor method instead.
Step-by-step
WE KNOW
nAT = 11, nGC = 9
FIND
primer length in bases (n), GC content (%GC), melting temperature (Tₘ)
Step 1 · Find primer length in bases (n)
Use n = nAT + nGC
Every base is one of A, T, G or C.
n
=
11
+
9
n
=
20
Step 2 · Find GC content (%GC)
Use %GC = 100 × nGC ÷ n
The G + C count as a percent of all the bases.
%GC
=
100
×
9
20
%GC
=
900
20
%GC = 45%
Step 3 · Find melting temperature (Tₘ)
Use Tₘ = 2 × nAT + 4 × nGC
Add 2 °C for each A or T and 4 °C for each G or C.
Tₘ = 2 × 11 + 4 × 9
Tₘ = 22 + 36
Tₘ = 58 °C → 20 bases is inside the 14 to 20 the rule is meant for.
Check
Put the numbers back in. Both sides should match.
20 = 11 + 9 ✓
45
=
100
×
9
20
✓
58 = 2 × 11 + 4 × 9 ✓
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