Hi all,
I'm having problems with my beta actin gene melting curve. In the past, the melting curves were working well (Fig. 1). Recently, I've faced with some problems (Fig2).
1- The y axis (fluorescence) shows different levels, even comparing the same sample (different technical replicates) - Pipettes are calibrated.
2 - The beginning of the curve are messy and high, comparing with my first figure. The primers are new.
Anyone have any idea what is the problem and can help me?
Thank you so much
Laurence Stuart Dawkins-Hall
- If you notice the scaling is different for the 2 examples, suggesting weaker signal for the output displaying more erratic peaks
- Taking into account this scaling representation, because you still only have 1 principal peak I would not be unduly concerned by such data
- However, you might want to run those products on agarose gels and just see if the issue is indeed S:N
- I think the data is usable: I would b more concerned if you had additional sharp peaks apart from the one shown
0 votes
0 thanks
Vinicius Richardi
The negative controls not amplified, Ct values were around 17-19.
0 votes
0 thanks
Oleg Suslov
Probably you introduced a new reagent for last PCRs. You can take ALL PCR components from other lab to see if it will improve the curves.
0 votes
0 thanks
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence
More Vinicius Richardi's questions See All
Similar questions and discussions