Two dissociation curve peaks in my standard curve - (Nov/08/2006 )
Hello,
I am hoping that maybe someone would be able to help me, or at least have heard of my problem.
I am attempting to do real-time using sybr green to determine copy number with a standard curve.
I started out with 18s (I would like to use it as my normalizing gene). I discovered that I have two peaks in my dissociation curve fairly close to each other. (2 degrees apart). Originally, it appeared as shoulders on my peak, so I did a different dissociation curve in which I took more data points and saw two clear peaks. One peak is slightly smaller than the other. I have run the samples out on a gel, and I was unable to detect two bands. No primer dimer. (my amplicon is ~250 bp). I have tried more concentrated agarose and I cannot get it very clear. I get a smear which I have been attributing to running the product too slow/too fast.
It is odd because I see the two peaks in my standard curve samples (amplicon inserted into a vector and then cut once to linearize it and cleaned up) as well as my samples.
I have tried different primers and saw the same two peaks. (I tried both slightly shifting my primers as well as completely different primers. All primers amplify a similar region because I am working in an Antarctic fish and we do not have the entire sequence).
I also tried a gradient to increase my annealing temperature. A two degree increase still had both bands. Any higher temperature and the reaction did not work.
I tried DMSO thinking it was due to secondary structure (some products base-pairing to form double stranded pieces while others fold on themselves?). I diluted DMSO 50:50 w/ water and tried different amounts of DMSO. Higher DMSO resulted in decreased pcr efficiency but did not get rid of the two peaks.
Things just got a lot wierder yesterday when I ran a few samples with VEGF primers (which also give me one band). I ended up with two peaks in my standard curve and one in my rxn with cDNA. (I think I only saw one peak because there was a lot less vegf in the cDNA than the Std. Curve and so I only saw the predominate peak).
Sorry this is so long, but I have tired a lot and given it a lot of thought.
If anyone knows anything PLEASE let me know!!!
KIM
I am hoping that maybe someone would be able to help me, or at least have heard of my problem.
I am attempting to do real-time using sybr green to determine copy number with a standard curve.
I started out with 18s (I would like to use it as my normalizing gene). I discovered that I have two peaks in my dissociation curve fairly close to each other. (2 degrees apart). Originally, it appeared as shoulders on my peak, so I did a different dissociation curve in which I took more data points and saw two clear peaks. One peak is slightly smaller than the other. I have run the samples out on a gel, and I was unable to detect two bands. No primer dimer. (my amplicon is ~250 bp). I have tried more concentrated agarose and I cannot get it very clear. I get a smear which I have been attributing to running the product too slow/too fast.
It is odd because I see the two peaks in my standard curve samples (amplicon inserted into a vector and then cut once to linearize it and cleaned up) as well as my samples.
I have tried different primers and saw the same two peaks. (I tried both slightly shifting my primers as well as completely different primers. All primers amplify a similar region because I am working in an Antarctic fish and we do not have the entire sequence).
I also tried a gradient to increase my annealing temperature. A two degree increase still had both bands. Any higher temperature and the reaction did not work.
I tried DMSO thinking it was due to secondary structure (some products base-pairing to form double stranded pieces while others fold on themselves?). I diluted DMSO 50:50 w/ water and tried different amounts of DMSO. Higher DMSO resulted in decreased pcr efficiency but did not get rid of the two peaks.
Things just got a lot wierder yesterday when I ran a few samples with VEGF primers (which also give me one band). I ended up with two peaks in my standard curve and one in my rxn with cDNA. (I think I only saw one peak because there was a lot less vegf in the cDNA than the Std. Curve and so I only saw the predominate peak).
Sorry this is so long, but I have tired a lot and given it a lot of thought.
If anyone knows anything PLEASE let me know!!!
KIM
rRNA genes are multicopy in the cell. I don't know why your standard curve shows the same problem though, and I think 18s is a typical gene to use.
Still, I suggest using a different housekeeping gene.