Most of the amplicon size in the realtime PCR are small, it can anneal and extend at 60 degree Celsius. Besides whether the product are completely extended or not doesn't make any impact in the quantification because realtime PCR quantified how much transcript number are available.

Real-time PCR buffer contain a detector dye mostly SYBR and passive dye to normalize the noise usually ROX, rest will same.

Dear Timothy

Standard PCR allows for the amplification of a specified region that will ultimately have to be assessed for success using some form of gel electrophoresis.  Real-time PCR uses an intercalating dye of sorts and allows for visualisation of amplification in 'real-time' as the PCR takes place.  There are a number of benefits to this, namely absolute and/ or relative quantification.  The informativeness of this, however, depends on experimental design and the inclusion of standard curves/ controls.

Regards,

There are no differences between buffers for conventional PCR or TaqMan real-time PCR except the addition of TaqMan probe to real-time PCR buffer.

About SYBR Green buffer - see discussion:

https://www.researchgate.net/post/Troubleshooting_homemade_SYBR_green_master_mix_preparation

I am wondering most of the conventional PCR is 3-step. It has an elongation step at 72 degree Celsius. But real time -PCR usually only has 2 step which has an annealing/elongation step at 60 degree Celsius. I am thinking what makes the difference between two.

Most of the amplicon size in the realtime PCR are small, it can anneal and extend at 60 degree Celsius. Besides whether the product are completely extended or not doesn't make any impact in the quantification because realtime PCR quantified how much transcript number are available.