Hi Woratit Woratit Intap

So let's check if I've understood the situation correctly.

You used to extraction methods to extract the genomic DNA from the same urine sample. Using that as the template, you got peaks at different temperatures.

Apart from having received different results for your target, looks like your control (the pink curve) is also acting differently between the two tests, am I right?

Also, have you got any info about the DNA content resulting from each of the extraction methods as well as their purity indexes?

Best,

N

Negar Fakhari thank you for your attention.

yes, l used the two different DNA extraction kits to extract genomic DNA from the same urine sample and using that as template in real-time pcr reaction. the pcr mixture and pcr condition these not different, but i got melt peak not the same location. obviously, peaks of DNA from kit A (blue) are lower than kit B (green). but control (pink) got different between the two test is right because one test is wild type (have a peak at about 92.5 degree C) and one test is carrier of thalassemia SEA type (have peaks at 92.5 and 87.5 degree C). although, peaks of DNA from kit A (blue) and kit B (green) are not the same location but they are same target gene that I want.

In previous study ( independent study of bachelor degree) were similar method used these DNA extraction kits to extract DNA from blood sample and reported as similar results but not detailed discussion. And also reported purity of DNA from both extraction kits using Spectrophotometry, DNA from kit A was 2.59 and kit B was 0.52.

So, I just want to known why melt peaks are the location different despite being the same target gene and the only difference is using the extraction kit?

DNA purity affects to shifted or not? or another reason

I apologize if my English is difficult to understand or impolite. Because I just learned to use it not long ago.

thank you

Hello,

Minimal ionic changes in the DNA extract will affect the master mix composition; therefore, detecting some displacement on melting peaks is quite normal. You should use two positive controls for each DNA purification method.

Woratit Intap

Many thanks for taking the time to give me more details.

Francesc Codony's suggestion might make the situation clearer, I guess.

Best,

N