Hi Shin,

As far as I know the data collection is performed in the extension step, since you want to get the curves representing the amplification through time. Are you using SYBR master mix or TaqMan probes? With SYBR, the data is collected from the fluorescence generated from the incorporation of SYBR into the newly synthesized double strand DNA, so the data is collected during the extension step; I don't think that collecting data at the annealing step will return any info back using SYBR, unless this is specifically specified by the master mix manufacturer. I don't really know how it works with TaqMan probes.

Nevertheless, check carefully the instructions for the qPCR master mix you are using. If the answer is not there, I would e-mail the manufacturer asking at which time point it is ideal to collect the data.

Hope it helps!

Best.

Catarina, thanks for the answer. I am using SYBR from Qiagen. I have used the same cycling protocol for TD-PCR and data acquisition is during the extension step. Below is the cycling protocol:

Real Time PCR Protocol:

Cycle 1: (1X)

Step 1: 94.0 °C for 03:00.

Cycle 2: (32X)

Step 1: 94.0 °C for 00:45.

Step 2: 50.0 °C for 00:30.

Increase set point temperature after cycle 2 by -0.5 °C

Step 3: 72.0 °C for 00:45.

Cycle 3: (20X)

Step 1: 94.0 °C for 00:45.

Step 2: 50.0 °C for 00:30.

Step 3: 72.0 °C for 00:45.

Data collection and real-time analysis enabled.

Cycle 4: (1X)

Step 1: 95.0 °C for 01:00.

Cycle 5: (1X)

Step 1: 55.0 °C for 01:00.

Cycle 6: (81X)

Step 1: 55.0 °C-95.0 °C for 00:10.

Increase set point temperature after cycle 2 by 0.5 °C

Melt curve data collection and analysis enabled.

After running SYBR 2-steps PCR, I ran the gel and it gave me unspecific bands. But with the same TD-PCR protocol, I am able to get specific desire band.

Conventional TD-PCR cycling protocol:

Cycle 1: (1X)

Step 1: 94.0 °C for 03:00.

Cycle 2: (32X)

Step 1: 94.0 °C for 00:45.

Step 2: 50.0 °C for 00:30.

Increase set point temperature after cycle 2 by -0.5 °C

Step 3: 72.0 °C for 00:45.

Cycle 3: (15X)

Step 1: 94.0 °C for 00:45.

Step 2: 50.0 °C for 00:30.

Step 3: 72.0 °C for 00:45.

Cycle 4: (1X)

Step 1: 72.0 °C for 05:00.

Cycle 5: (1X)

Step 1: 4.0 °C hold.

Hi Shin,

Sorry, I never did TD-PCR, the program I use is different from yours then. But I got the impression the data collection is still in the extension step?

It is okay. Yup. Normally it will be during the extension step. Anyway, thanks for the advice. All the best ^^

Guys, I managed to optimised the real time TD-PCR protocol. Thanks for the advice and suggestions..

When you will collect data you must consider the conditions in which the fluorescence will be accumulated at the maximum.

So for both SYBR GReen and Hydrolysis probes (NOT TAQMAN, the use of this term is advised against by MIQE, Minimum Information for publication of Quantitative Real-Time PCR Experiments, Bustin 2009), signal is optimal at the end of the elongation phase. Then all DNA is synthesied and loaded with SYBR Green, or hydrolysed completely (hydrolysis probes). Molecular beacons however, must be measured before elongation phase starts. These detach from the single stranded amplicon when temperature rises to elongation temperature.

Data aqcuisition must take place during each cycle and begon with the first cycle of the TD menue.

Hi, Elizabeth. Thanks for the answer. I am not very understand about your explanation. Is it possible for you to give an example like cycling protocol?