Help with melt curve

Use this category for questions related to various quantitative PCR methods, including real time PCR, reverse transcription (RT)/PCR, qPCR, etc. For general PCR methods, continue to use the DNA/General PCR forum

Moderators: r.rosati, Suzanne

Help with melt curve

Postby jtan091 » May 19 2017 9:20 am

Hi,

I am very new to qPCR. The problem I face is regarding the melt curve of 1 set of primers.
I performed a trial run and the melt curve gave only 1 peak.
K14 1.jpg

However, the next day I ran the real analysis using the same samples, and the melt curve gave multiple peaks.
K14 2 1.jpg

This is observed in all replicates from 3 samples. All the other primers are good, even the NTC for this primer is good (no peak at all).

I have ruled out contamination from the reagents as well as the template. I also took cautionary steps to reduce pipetting errors; I had some problems regarding pipetting errors in previous runs. I'm really lost as to what is the cause of this observation. It would be nice if you could suggest some reasons and how could I prevent this from happening again.

Thank you :)
You do not have the required permissions to view the files attached to this post.
jtan091
newcomer
newcomer
 
Posts: 1
Joined: Apr 23 2017 8:35 pm

Return to Real-Time qPCR/qRT-PCR Methods

Who is online

Users browsing this forum: No registered users and 1 guest