Why my RNA absorbance reading shown negative? - (Sep/05/2007 )
Hi..I had extracted some rna from some water samples in which i needed to know how many concentration they are. When I used the spectometry to read it..it shown negative reading for both 260nm nand 280 nm . I tried to do it twice n blank it twice to ensure that i do it right..but yet i still couldn't. Can anyone help?
well quite surprising.
Check a preparation of plasmid DNA your sure about. It's less fragile and will allow to check your lamp too
reason 2 : you may have a dirty cuvette. Wash it thouroughly with water and do not rinse with etoh (will false the zero, particulary at 230nm).
Then put back your sample less diluted.(maybe your sample is not very concentrated too)
Oh..I am sure that I washed the cuvette throughly and the dilution I am doing is 50 times..Maybe I should do the 10 times dilution? but to tell you frankly..I am also lack of samples to continue for my hybridization work. Any more suggestion? Your help is very much appreciated. Thanks..Fred..
you may try a nanodrop if there's one close to you, which use less amount of solution.
I don't know how to do hybridisation without knowing the concentration. So at this point your exp is stopped. I'm sincerely sorry for you.
HAve you check it with normal gel electrophoresis? Maybe you dont even have any RNA at all?
Negative OD readings when spec'ing DNA or RNA means you have Ethanol contamination in your prep! To ultamitely check this mix with loading buffer and load into Gel. If your volume floats out of the well you do have ethanol contamination which was carried over from your wash step.
Yea.. maybe RNase contamination. Better check that or else you wouldnt get any readings at all.
Oh..well...i will try to check it out. I also afraid of ethanol contamination..but the gel electrophoresis shown band. So can anyone help here? Really headache..and desperate.
what is the estimated concentration ? if it's very diluted (ie low concentrated) it is tough to have a correct measurement (although it's not impossible)
also may you estimate your concentration regarding your gel ?
I think you have a low concentration of RNA. Spectrophotometer in not very sensitive and have many chances of errors. If it’s not calibrates and or your cuvettes are not ok and the RNA is very diluted or have some ethanol contamination, you won’t be able to measured.