Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix - Manual (Page 23)

| 17 SsoAdvanced ™ Universal SYBR ® Green Supermix Instruction Manual | 17
If your melt profiles exhibit additional peaks at higher melting temperatures than your
product of interest (see Figure 14), this is most likely due to nonspecific binding of the
primer(s). To correct, please consider the following:
Table 5. Primer matrix.
Forward Primer, nM
Reverse Primer, nM 100 150 200
100 100/100 150/100 200/100
150 100/150 150/150 200/150
200 100/200 150/200 200/200
Fig. 14. Mis-priming event around 90ºC.
Temperature, °C
65 70 75 80 85
–d
(R
FU
)/d
t
90 95
3,500
3,000
2,500
2,000
1,500
1,000
500
0
1. Evaluate the assay design by following the bioinformatics workflow outlined in the beginning
of this manual (page 3). This will help ensure that the primers are highly specific to your
target of interest, and no other target region(s).
2. Perform a temperature gradient to determine the optimal annealing temperature of the
primers. Load your plate with the same reaction setup and sample for each primer set in a
column format so that you can evaluate the annealing temperatures. Set the gradient 10ºC
above and 6ºC below the calculated annealing temperature to ensure a proper temperature
range is covered. Choose the best temperature based on the melt profiles (no extra peaks),
keeping in mind that lower temperatures may reduce specificity and higher temperatures
may reduce primer binding efficiency.
Fig. 15. Temperature gradient layout.
Primer
Set 1
Primer
Set 2
Primer
Set 3
Primer
Set 4
10°C above
Annealing T
m
= 60°C
6°C below
A 70.0
B 68.9
C 66.9
D 64.0
E 59.8
F 57.1
G 55.2
H 54.0