Hi, everyone!
I was doing a flurecence test recently by using DISC3（5）which can insert into membrane to detecte the change of membrane potential of bacteria, and this is my first time to do a flurecence related experiment . However, except at the first time that the result of the kinetic curve(detect every 5 minutes for 1 hour) is smooth, the curves of the follwing experiments are not, they fluctuated very much, see the file below. I don't know why, could anyone give my some advice, thanks!
PS: I tried DISC3（5）at a final concentration of 0.4μM and 0.04μM in bacterial suspension of 10^7 CFU/ml.
Perhaps the problem is a lack of signal. Were the wavelength and height settings optimized? For DISC3(5) the excitation and emission peaks are at 651 and 675 nm, according tho the Molecular Probes catalog.
Another problem could high background. Are you using black plates to minimize background? Are your cells fluorescent at the wavelength settings.
Adam B Shapiro Thank you for your advice. the wavelength I used were 622/670 and 500/707nm, one from an article and the other from the Commodity description, and now you tell me 651 and 675 nm, It's a little hard to chose.
I used black and clear bottom plate , is that right?
Since at the first time the curve is smooth , could that fluctuation comes from the repeated freezing-and-thawing?
Freezing and thawing of a solution causes separation of the solutes from the ice. If the thawed solution is not mixed, you could end up with a lower concentration of the solute than expected.
Some fluorescent dyes are prone to photobleaching when exposed to light. Care should be taken to avoid unnecessary light exposure.
I would suggest using a solid black plate unless you need to be able to see through the bottom of the plate.
622/670 should be acceptable, if not quite optimal.
- Badges
- Science topic
- Similar topics
- Filing