I was making experiment on tagging natural DNA with fluroscent dNTPS . I tried to read the incorporation via nanodrop because it has visible spectrum . What i found is two large curve lines with peaks at the UV range and a faint lines at visible range.
The interesting think is that my control nonfluroscent DNA product had only one curve line at UV wave length . I need to know is there any better way to ensure that incorporation was successful so as to go to reading step. Regarding this part of reading this fluroscent DNA using sequencers . I need to be read only without the known steps of amplification and reading by synthesis because it is already enough in amount and also tagged .
It looks like the absorbance in the UV is saturating - an absorbance of 2.2 is a transmission of
Many thanks Mr.Byrne .
What about the second graph that nominated with fluroscent DNA which is an experiment that used two fluroscent nucleotides with different exc/em range but are close to each other. Is this graph mean incorporation is done or not ?
To be sure -you should dilute your product so that the max absorbance is the same as the DNA, and you should have an absorbance spectrum of your dNTPS . To first order, your absorption should be a sum of the two spectra.
And then you should do fluorescence.
My initial dyes natural colour is yelllow and orange solution so i expect to reflect light at 400 to 480 , beside that they are fluroscent and get exited at waves of 490 and 505 . Is this will explain flattening of the curves at 400 nm as my spcetrophptometer doesnot read emission waves
You are measuring using absorbance - which is ok.
But this initially measures Transmission = Isample/Ireference, and then converts to Abs = -log(T).
If your sample is highly absorbing, (A=2 means a transmission of 1%), then you are dividing almost zero by your reference, and calculating the log. This can give incorrect results, particularly at the UV end of the spectrum, where there is not very much light anyway.
Dilute the sample by a factor of ~10 and take the spectrum again.
I will diluate my control DNA as you said Mr.Byrne .
Thanks you very much for your helpful advices
I went to measure the DNA purity as will to ensure that i had DNA sample using nanodrop DNA setting in the program, also to ensure that just above reads that i had on UV- VIS measure is not just the free labelled dNTPS .
- Badges
- Science topic
- Similar topics
- Chemistry
- Sulfites
- Inorganic Chemistry
- Inorganic Chemicals
- Salts