Hi,
I am working on gene expression, and want to ask about 2 issues ...
I am using SYBR green, but I am confused because each kit has its own specific Ta, while the primer itself has a different one, so I want to know which one to work with?
And also which is better, 2 step (annealing with extension) - or 3 step- reaction??
Thanks in advance.
Hi Noha Abu-Nasra
Usually, follow the kit guidelines for primer design and qPCR. For example, Quantifast Kit (Qiagen), recommend a two step protocol with 60ºC anneling/extention step even for primers with Tm below 60ºC.
For primers, design them with 60-65ºC Tm and they should work properly with most two step protocol qPCR kits. A good software to design qPCR primers is primerquest tool (https://www.idtdna.com/Primerquest/Home/Index).
Best,
Jacó
Hello
If the Ta of your primers has a big difference with the kits recommendation, you may try primer Ta and see what happen. Two-step is recommended when you are using a probe, 3-step works fine with SYBR. BTW what kit are you using? Usually companies do not reveal primer sequence in their commercial kits!
Good luck
3 steps are better, since the optimum temperature is being used while the elongation is taking place. The aligning temperature, generally, depends on primer Itself, since, depending on its sequence, the aligning temperature is going to change. SYBR green works like that
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