useing pcr product as template in qPCR with spesific tm but no band on gel?

[s-vet]

i use pcr product as template in real time pcr to get the abslute stundard curve and then calculate the amount of my samples(genomic dna). always i have band for my samples but i have no band for stundards! but specific tm and peak for both!!
the starting point of diluation serial have E7 copy number of target.
another problem is high efficiency in stundard curev or low effiinecy ! i can't get a good result !
anyone know if there is any diffrence in pcr condition between genomic dna and pcr product!
thaks for any help

[obama]

In my experience Never use PCR product as template in most of the time it won't work. Solution Use TA TOPO cloning kit and clone ur pcr product into TA vector, then use it as template for ur pcr standards.

And am not able to understand your second part of question regarding efficiency.

[s-vet]

thanks you
i have no expersience , could you pls help me more about working with this kit and makeing stundards? i dont know what should I do
best regards

[obama]

Originally Posted by s-vet

thanks you
i have no expersience , could you pls help me more about working with this kit and makeing stundards? i dont know what should I do
best regards

just type TA TOPO cloning kit in Google search you will get information regarding it , I think it is from invitrogen. (you can find detail explanation how to do this in there manaual). Once you done with cloning then isolate the Plasmid which has your pcr product now use this plasmid as your standard.


If you are not able to buy this kit for any reason. Then one more way is to design a primer with restriction site then amplify your pcr product then clone in to plasmid using the same restriction site. (Hope you know about cloning) . Once it is done then you can use that plasmid as your standard template.

[obama]

here is link for the product from Invitrogen
[Only registered users see links. ]

[butters]

I have used PCR product as template in realtime, no problem. From what I gather most likely are using too high concentration. please check that your amplification plot is it an enlongated n-shape? and more probably just around your threshold line.

please supply your starting DNA concentration and your PCR product size. I will see what I can do.