Hoechst 33258 problems! - DNA quantification, fluorescence (Apr/11/2005 )
I'm trying to make a standard DNA-koncentration curve using Hoechst 33258 in a fluorometric assay. But its not working the way I want it to work...
I cant repeat the curves made! Most troublesome, the fluorescence value for the blank varies!
Have someone encountered the same problem or has a possible explaination/solution, I would be really grateful!
// josefin
I would recommend creating a new standard curve for every batch of samples you'd like to quantify. You would expect some deviation, but it shouldn't be extreme.
As far as the blank - I believe the fluorometer is very sensitive to residual fluorescence from the cuvette (maybe from a prior sample). Either way, the protocol for our instrument requires blanking the machine prior to each reading with buffer (containing free Hoechst) just prior to the addition of sample.
One final question - were you using the same stock of calf thymus DNA to create your standard curves?
As far as the blank - I believe the fluorometer is very sensitive to residual fluorescence from the cuvette (maybe from a prior sample). Either way, the protocol for our instrument requires blanking the machine prior to each reading with buffer (containing free Hoechst) just prior to the addition of sample.
Hi!
I'm just a little to lazy to make a new curve every time... And sometimes I only have a few DNA-samples, it wouldn't be worth the effort. It does work with PicoGreen (which I tried as an alternative to Hoechst, now when things went so badly as described).
I'm using microtiter plates (a new one everytime), so I should have none residual sample.
As for the blanking procedure: our fluorometer does only allow blanking of the well A1. This, of course, is not very practical... So unfortunatly, no blanking can be done. I do, however, always have a blank sample in my curves. The zero vlaue does indeed vary, but also the slope of the curve.
Yes, and a rather freshly made DNA too.
I appreciate the help, but I think I simply have to give up on Hoechst 33258. I have tried so many things, but nothing helps. And now I even poured out the TNE-buffer to prevent me from doing any more experiments (else I have trouble letting go)
// josefin