Hi Hafiz,

There are two or 3 temperatures in a PCR cycle. The size is not that important, but the primers are.

Dissociation:

For any DNA strand a dissociation temperature of 94 or 95C is sufficient. There is an initial hold at this temperature, usually about three minutes. However if you are using an Taq enzyme that requires heat activation this initial hold may need to be longer, check with your reagent manufacturer.

After the initial high temperature hold begin cycling with 30 seconds at the dissociation temperature.

Annealing

The annealing temperature is usually set about 5C below the primer melting temperature. you can calculate that using a number of online tools. (one I like is at http://biotools.nubic.northwestern.edu/OligoCalc.html

for almost all PCR reaction you can use the sites default settings for salt and primer concentration and it will work fine. If your two primer melting temperatures differ by more than a few degrees you can make a shorter version of the one with the higher annealing temperature to make them similar. This is a rather rapid reaction given the relatively high concentration of primers in a PCR reaction.

Extension:

extension temperature (can be the same the same as annealing, but often is not). Optimal extension temperature for Taq is 72C. I would give it a 90 second extension. longer extension times can help if you have a long product or a difficult to read through sequence.

These are typical conditions, I am not sure how abundant your target is in your samples which would impact the number of cycles to run, but 35 is a good starting point.

I hope this was helpful.

Frank

Hi Hafiz Muhammad Asif Langah , as Frank R Burns explained, the PCR cycle includes 3 steps:

1-Denaturation: 94-95°C

2-Annealing (hybridization): primers Tm temperature dependant

3-Extension: 68-72°C

The size of the product is important to determine the extension time: Typically 1min/kb for Taq DNA Polymerase.

This link could be useful: https://www.thermofisher.com/ma/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-cycling-considerations.html

You'd have to tell us something about your primers to know what annealing temp to use.

In general

1. 95* 5:00

2. 95* 0:30

3. [48-65*] 0:30

4. 72* 1:00

5. Repeat 2-4 35X

6. 72* 5:00

7. end

You'll need to customize depending on your polymerase of choice, most commercial ones require the 5:00 initial hold to activate the enzyme. The general rule is 1:00 per kb for extension, can be faster with a high copy template, longer for a difficult template. Number of cycles can vary too.

If you don't have information about the primers, use a gradient of annealing temps & you can determine which is the best.

Good luck!