User:Nadiezda Fernandez-Oropeza/Notebook/Notebook/2011/05/25

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Progress on the Motility Assay

 * This entry contains some personal remarks on the process of completing the motility assay.


 * 1) First try: No microtubules =(
 * 2) Second try: In the second trial, I got some microtubules, but really big fluorescent clusters. This might have been due to the fact that I used old aliquots of the 29% Rhodamine-labeled tubulin suspension.The following are some of the things I had to learn the hard way:
 * 3) *DO NOT leave the thermal cycler, the mercury lamp or the camera turned on!!!!!!!
 * 4) *Make aliquots of the antifade.
 * 5) *Taxol takes up to 5 minutes to defrost then you should give yourself some reasonable time before preparing the Taxol in PEM solution.
 * 6) *Ideally, the motility solution should be prepared in the 5 minutes of incubation of the Kinesin with the PEM-αA in the flow cell.
 * 7) *Try to add the PEM –Glu almost at the end.
 * 8) On my third trial and four trials, I got a lot of microtubules. The sample was way more populated than expected. I really don’t know why this is, I have the suspicion that it has to do with where on the tube I collected the volume of tubulin with Taxol in PEM. Discussing this with Andy, he said that it shouldn’t really matter. Furthermore, the image didn’t have a lot of contrast between the microtubules and the background. This might have to do with the way I focused the sample or maybe just with the fact that my sample was really overpopulated.
 * 9) On my fifth trial, the number of microtubules was more reasonable, unfortunately, I still have the contrast problem.


 * }