User:Anthony Salvagno/Notebook/Research/2010/04/21/Quantum Dot Tryouts
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I will try and work with the Qdots in both regular water and heavy water just to see what it is like. If I can make a movie I will do just that. Here is what I will do:
- I won't use clean glass
- I will use 2 chambered samples
- In each chamber I will flow the same wavelength QD but in one chamber I will use the incubation buffer I was supplied with. In the other I will just dilute with water.
- One sample will be heavy water the other will be regular water.
Preparing Qdot® Streptavidin Conjugate: Do not vortex the Qdot® streptavidin conjugate vial. Prepare the required amount of the diluted Qdot® streptavidin conjugate needed for the experiment on the day of use. You will need 40–200 μL Qdot® streptavidin conjugate per coverslip depending on your protocol and the type of humidity chamber you use.
- 1.1 Centrifuge the Qdot® streptavidin conjugate vial at 5000 × g for 3 minutes prior to use. Use only the supernatant and discard any pellet.
- 1.2 Dilute the conjugate by adding 2 μL of the stock (1 μM) conjugate to 100 μL Secondary incubation buffer immediately prior to use to obtain the Qdot® streptavidin conjugate concentration of 20 nM (you may use between 10 nM and 40 nM Qdot® streptavidin conjugate final concentration).
- 1.3 Use the diluted Qdot® streptavidin conjugate immediately for the current experiment. Do not store any diluted Qdot® streptavidin conjugate.
I mixed 2ul of each QD using the above protocol. Then I mixed 1ul of each QD with 50ul of D20 for my heavy water solution. I then flowed 10ul of each solution (4 solutions total) into sample chambers. I'm letting it sit for a few minutes and then I will seal with nail polish.
I don't know why, but the live feed wasn't working at all. I tried to look at a very bright piece of tape and the screen was just black. Yes, I even checked to make sure it was in camera mode on the microscope. Anyways I'll just have to report my findings by eye.
First I'd like to say that visualizing quantum dots is cool. Very cool. Ok now that's out of the way. My first method of visualization failed. I think there were too many QDs in solution. After Koch's suggestion I then flowed 10ul of QDs and then washed several times with Water and D2O. I just used one color QD this time and had two samples. One sample was washed with water and the other with heavy water (just to clarify that I didn't wash with both in each sample).
I could easily visualize the QDs in regular water and had trouble seeing the heavy water ones. I didn't really notice if the blinking was different, although I suspect that in heavy water they were more off. It also seemed like there was more background fluorescence in the heavy water sample but that could just be because there are more free QDs. Also I don't notice any sort of color. This would be bad for project lambda if this is the case.
Ok I just checked the heavy water sample again and it magically fixed itself. The background isn't as bright (photobleaching?) and it seems like the blinking rate is increased from the regular water. I'm going to play around more and if I notice anything new I'll report it.
This is a picture of a mass of Qdots with these settings from the live feed:
- exposure 0.5
- gain 200
I don't get why it needs to be so high to see the dots? Oh well.
- Steve Koch 18:29, 22 April 2010 (EDT):Ant finally discovered that the ND 2.0 filter was still on! After taking off, we got very nice movies. Ant will upload them to see side-by-side soon.