Crazy dissociation curve, please help ! - Strange peaks in dissociation curve (Jun/16/2008 )
Hi, I did a lot of QPCR before, they worked perfectly. But since I moved to this new lab, everything changed from reagents to machine. I've been doing lots of troubleshootings recently trying to figure out what in my real-time PCR went wrong. Two of the most annoying things I found in my results are strange peaks on dissociation curve and different plateau levels. To make things easier to understand, the two pictures linked are reactions using the same primer sets and 1:3 serial diluted cDNA templates. By the way, I have run some of the sample in agarose gel and only one band showed up.
http://www.fotothing.com/photos/6b8/6b8752...f684038_051.jpg
http://www.fotothing.com/photos/883/883734...3d37af3_09d.jpg
I am thinking of either my template concentration is too low or there is some contamination occurs in my experiments.
Any advice or suggestions will be highly appreciated, thanks!
Any advice or suggestions will be highly appreciated, thanks!
Based on the amplification curve, the first thing is increase the input amount of templates. Secondly, supposed you run NTC, adjust the background with NTC.
Any advice or suggestions will be highly appreciated, thanks!
Based on the amplification curve, the first thing is increase the input amount of templates. Secondly, supposed you run NTC, adjust the background with NTC.
Yes, I did NTC control, see the links for pictures. But I don't know how to adjust the background with NTC, is there a particular function in the program? - I am using AB SDS software v1.4. Thanks.
http://www.fotothing.com/photos/98e/98e21f...26bfcc4_93c.jpg
http://www.fotothing.com/photos/2f8/2f8eb0...ab558bc_760.jpg
Have you run all your samples on gel? You said some..
Also, have you tried to increase your Tm?
Also, have you tried to increase your Tm?
I had run the first 3 samples in the dilution series and loaded 8 ul out of 25 ul reactions on a 2% gel. I saw clear strong signals, just one band with expected size.
The Tm of my primers is 60C and I used 55C for annealing temp. I can try 57 or 58 next time to see if those peaks go away.
Thanks!
Also, have you tried to increase your Tm?
I had run the first 3 samples in the dilution series and loaded 8 ul out of 25 ul reactions on a 2% gel. I saw clear strong signals, just one band with expected size.
The Tm of my primers is 60C and I used 55C for annealing temp. I can try 57 or 58 next time to see if those peaks go away.
Thanks!
Hi, I just figured out the reason of my QPCR failures. It's the freaking tubes! Someone ordered a box of tubes assuming they are RNase-Free, but they are NOT! I changed sterile tubes and the peaks now look beautiful!
Thanks you guys.
Also, have you tried to increase your Tm?
I had run the first 3 samples in the dilution series and loaded 8 ul out of 25 ul reactions on a 2% gel. I saw clear strong signals, just one band with expected size.
The Tm of my primers is 60C and I used 55C for annealing temp. I can try 57 or 58 next time to see if those peaks go away.
Thanks!
Hi, I just figured out the reason of my QPCR failures. It's the freaking tubes! Someone ordered a box of tubes assuming they are RNase-Free, but they are NOT! I changed sterile tubes and the peaks now look beautiful!
Thanks you guys.
First, thanks wangloada for sharing the solution with us!
Next, ABI software does not allow you to correct for NTC.