Frustration

Well, I was expecting this one to work but for some reason it didn't. Again, I don't know why. I used less cells this time which probably wasn't the problem. This time, the problem was that I got contamination again from somewhere. I still have no idea where it's coming from. Nothing should be in my final sample except the DNA I'm interested in. It doesn't make sense. It's very very frustrating.

In the hopes of eliminating any contamination, we're going to add another purification step. This step involves using nickel beads, which are very similar to the avidin beads I've been using. The principle idea is the same. The nickel beads will bind to the tagged ubiquitin and everything else won't. Then after we wash the beads a couple times, we break the bonds between the nickel beads and the tagged ubiquitin and capture whatever has the tagged ubiquitin protein. Then I'll take that sample and apply the avidin beads like I have been doing. We're hoping that adding a second purification step will eliminate any contamination. We'll probably get less yield as a result of doing another purification step but we should have enough DNA to do the qPCR and get decent results.

That's the theory at least. But nothing much else has worked for me recently so I've learned not to get my hopes up.

Finally Got Some Good Results

The double depletions did the trick. I got the results I expected with one of the siRNAs I was using. However, the new one I was using, the important one, didn't show the same results. I think I know why this is and I don't think it's a major problem so the results from this experiment are good news. Basically, my mistake was using too many cells. If I use too many cells, my siRNA will basically get diluted and not work as well as it should if I use less cells. So the only thing I have to do is use less cells next time and I should see the result I'm hoping for. Things are finally going in the right direction.