I’m doing real time PCR with pepper samples. I’m trying primer concentrations. The Ct values are very low and the technical replicates are quite different. When the amplification arrives at the plateau phase, the fluorescence declines. We are sure the primers are working well because PCR was carried out and the results were successful.
Can anyone explain the reason for these real time results?
Attached you'll find our graphs.
Many thanks
Thanks Steve...yes to the SYBR technology.
Normally we first make the primers concentration and then with the correct one we check for the efficiency. So we are in the first step.
It's the first time we work with pepper and the primers were designed based on tomato sequences.
Wich could be the factor that produce the excessive excitation? Should we dilute the cDNA?
We did a cDNA dilution comprobation and with a 1/10 dilution seems to be ok.
what is the product size which was amplified with PCR? I think Product size may getting problem. Actually if we will work with SYBR dye we should take amplicon size ranges between 100 bp to 120 bp. If our amplicon size is more then there is more consumption of SYBR dye and eventually we will get fluorescence in declining condition at latter bcz this is a double stranded binding dye. So take care with your amplicon size.
Regarding to errors between technical replicates, this is due to pipetting errors.
And other thing is that whether you checked melting curve. Is it giving single peak to particular primer?
best of luck
Hi, I'm working with Consuelo in pepper...or better fighting with it, ;)
Thank you both of you for your help!
The product size are all around 100pb. We check it in a normal pcr.
Melting curve are all single peak.
Now we are going to check if maybe we have some inhibition from the DNase treatment or from the RNA extraction.
You wrote you checked for specificity / primers working with PCR. Did you switch from one polymerase to another, as in, different products/producers in reg.PCR / qPCR? This usually shifts reaction optimum some, too! Make sure in the qPCR, that this switch did not affect product specificity by HRM post qPCR / gel electrophoresis.
Since cDNA dilution enhances your qPCR, you're very likely experiencing some inhbition indeed.
The decline may be caused by software data interpretation. If you turn off base line correction you may see that you have drift on the initial baseline. The software corrects the whole curve.
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