My team and I want to do HRM (high resolution melting) for analyse change between uracile and citokine by construction of dissociation curve in ABI (Applied Biosystem) 7300 real time PCR system.
Dissociation curve constructed by ABI 7300 system is the same quality like HRM dissociation curve constructed by a more new version of real time PCR system?
Can I use with ABI 7300 system the LCG Sybr Green?
Can I use ABI 7300 system the Master Mix LCG Sybr Green?
Can I use with ABI 7300 system the software to analyse the HRM?
Hello, Evangelina
You need a special software to analyse HRM with ABI 7300 real time PCR system. You can use either LCGreen or SYBR dye but there is no LCG Sybr Green dye. But LCGreen is fully saturated and better than SYBR for HRM analysis. You can get this information from Idaho technology (http://biofiredefense.com/lightscanner/). I have been using LightScanner (HRM instrument) to detect mutation ans SNP of various genes. You can also see my papers on my profile. You cannot use ABI 7300 system software to analyse HRM because it is mainly for Real Time PCR. Since HRM needs at least one control to compare the test samples. But if you still want to use HRM with 7300 system, you need this software
http://www.appliedbiosystems.com/absite/us/en/home/support/software/real-time-pcr/hrm.html
But I recommend you to do HRM with LightScanner instrument and software for better result.
Good luck.
Hi Evangelina!
ABI 7300 and 7500 Real Time PCR systems do NOT perform HRM analysis, so you should avoid to use this systems for HRM studies. However, you have the AB 7500 Real time Fast system that is a version of Applied Biosystems suitable for HRM analysis. To perform an HRM analysis you need basically an adequate fluorophore (e.g. Syto, do NOT use Sybr green, the fluorophore must saturate the DNA molecule and Sybr green lacks this property), an adequate system (e.g. AB 7500 fast, Eco real time of Ilumina, etc..), and an adequate software (that comes with the machine that you use).
I hope i have been useful,
good luck!!
Sergio
Thanks a lot for your answers (Banyar Than Naing and Sergio Espinola).
I understand very well is not possible to do HRM in my ABI 7300 system, because do not use fluorophores and software for this test.
Well may be in a future we buy an advanced RT-qPCR system like AB 7500 or Fast System.
Evangelina
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