Dear Sirs, I am trying to do multiplex real-time PCR with SYBR Green. I had 4 different pcr products. But the difference of melting temperature of products are not different. At least by the online melting temperature analysis software. I tried doing duplex and triplex but the curves are not resolving, though monoplex are resolvable. How can these be made resolvable?
the monoplex temperatures are 82.4 83.1 83.3.
Are you using High resolution melting post PCR analysis to determine the melting temperature of the products and in that way discriminate several products in a multiplex SyBR GREEN qPCR?
In my experience with HRM, °Tm depends on the size and GC% of the product, so you could try to design primers as if you were designing primers for multiplex endpoint PCR (different sizes of products and products no bigger than 350bp for SyBR Green qPCR application) and then analize the different sequences of the expected products using uMelt to predict the different °Tm (https://www.dna.utah.edu/umelt/umb.php).
Dear Alejandro. Thank you. I am not using HRM. Using about 1 to 0.5C per step. If i use 0.1 or 0.05 the curves are too wavy to use. Will making big sized primers (>150bp) still keep the pcr reaction efficient. But that is a very good idea if I only want to see the end product.
Using SyBR Green, you can design primers for products until 350bp or no more than 300bp, but it's possible that even 30-50bp of difference in the different products, you could get enough different °Tm; however, as I told you before, °Tm depends not only on size product, but also GC%, so you could try to produce different products with sizes and also different GC% (let's say 30, 40, and 50%).
Furthermore, to get good discrimination in the °Tm peaks (using predictions from uMelt) try to get not less than 1.5-2°C between each °Tm (i.e. 76°C, 78°C, 80°C, 82°C). In the practice it could be hard to get this; but this is one of the ways that I've realize to discriminate different products using °Tm of the products.
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