Assuming that we have a good negative control, and the right melting temperature, what can cause very low fluorescence level of the melting curve?
Thank you for the answer Dear Govinda. In my case, the negative control does not have any peak, and when I checked the products on agarose gel everything was perfect, I mean, only the positive controls had amplification. The amplification had the right size and primer dimers were apparently absent! That's why I am intrigued on why the fluorescence peak is so low!
Next week I expect to try new Sybr green Mix, new machine and even new samples! If nothing changes, new primers. I just wondered if there might exist other reasons for having peaks with low fluorescence!
hi Marques
from ur discussion I understand that there s a problem with ur syber green to confirm completely u can set up a reaction with the kit control primers and template DNA (provided with most taq pol kits/reagents irrespective of amplicon size)...as u stated u could see products in gel but no fluorescence which makes to believe that the taq poly n other reagents in the master mix r fine n not the sybr green so buy/borrow a fresh aliqot of sybr green n try again...
All the best !!
Are you running the reactions until the amplification reaches the plateau phase? You don't have to do that to get the Cq value, but generally more product will get you more signal. If you are reaching plateau and still have little fluorescence, it could be your sybr green is not fresh, as mentioned above. If you are limiting your primer concentration, you may simply run out of primer before you reach a very high fluorescence, this is also true of any other reagent like dNTPs. Of course, using a smaller reaction volume, you will have less of every reagent, and you will have less fluorescence, eg) I had less fluorescence when using 12 uL reactions than when I did 25 uL reactions.
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