User:Steven J. Koch/Notebook/Kochlab/2008/07/17/HIV Reverse Transcriptase DNA Tethering
Steve Koch 14:40, 26 May 2009 (EDT): I am copying the below information from my private wiki notebook which I used back in those days.
Weighed out catalase and glucose oxidase, 4.9 mg of catalase and 9.8 mg of glucose
oxidase. We put the powder into 15 ml falcon tubes, and then addded the appropriate
amount of BRB80 (moderately chilled) and mixed by pipetting up and down. This was
6.125 ml for catalase and 4.9 ml for GOD. We then made 1 ml aliquots which are now
freezing in -20C in the box that was originally for antidig aliquots
Thinking about HIV RT
According to User:Steven J. Koch/Notebook/Kochlab/2008/07/16/investigating hiv RT, The HIV RT is about 8000 U / mg for 1.2 mg / ml (these are numbers she found from old preps, before information was proprietary...her name was Gretchen)
The RT I was using in grad school was estimated 4.3 micromolar, and I would use 1:100 dilutions of it. According to what Larry found, the heterodimer is 117 kd. To convert 4.3 micromolar to mg / ml ... 0.5 g / l which is 0.5 mg / ml.
10 ul of GOD 10 ul of CAT 5 ul of BME
Preparing next sample
10 ul 10x YOYO1 (from yesterday) 1 ul lambda DNA stock 0.5 ul RT (Ambion) 85 ul TE buffer
Then split into two tubes, and add 1.25 ul of cocktail + 0.5 ul dextrose to ONE of the tubes (which had received 48 ul of the above mixture).
Steven J. Koch 01:39, 18 July 2008 (UTC):The above sample was successful, and we EASILY saw tons of end-tethered DNA. In my opinion, the dominant feature of the sample was something that appeared like a bright dot stuck to the surface, then a wiggly segment, with a bright dot on the other end in solution. Pat and I thought this would make sense if the DNA was repelled from the surface, so the farther away from surface, the easier for the DNA to coil. However, explaining the dot at the fixed end is more difficult. All of this seemed very familiar to me, so I am thinking that it is exactly what I was seeing a number of years ago, when I took those old notes were were following We did not see tons of "guitar strings", but we did see a few, and even more prevalent than that were "jump ropes" which I also saw a number of years ago. Either at this sample, or the next one, we have video where Linh noticed a "slinky DNA." I am pretty sure our first video from this sample showed very cool stretching / recoiling / detaching of the DNA. It is very clearly better microscopy than with the nanoletters paper, and I think the video is even good enough to fairly represent what you can see with your naked eyes, which is great. Larry had some Murphy's law action going with his video acquisition, but even then it was WAY more easy to use than the Solis crap, and it was essential for our work today -- thanks Larry!
To wash the samples, I prepared "1x YOYO1 + antifade", by mixing 90 ul TE buffer + 10 ul 10x YOYO1 + 2.5 ul antifade cocktail (above). The antifade was an afterthought, so I think that is why the total volume was probably 102.5 (no big deal). I used this "1X YOYO1" with the later diluted DNA.
Preparing dilute samples
Steven J. Koch 01:45, 18 July 2008 (UTC):We decided that the samples were way too concentrated, so we decided to try diluting the samples. I am pretty I did this for the first diluted sample:
18 ul 1X YOYO-1 2 ul "no antifade" DNA mixture from above
For the latter diluted DNA (which I think I used for all subsequent samples), I am pretty sure I did the same as above, except I used 2 ul of "w/ antifade" DNA instead of no-antifade. This probably doesn't matter too much (whether i used w/ or w/o 10x concentrated DNA).
First diluted result
Steven J. Koch 01:48, 18 July 2008 (UTC):The diluted sample was much easier to view, and we got good video. The background was dimmer, so that you could see the end-tethered DNA better. We flowed again with this sample (washing with 1x YOYO1 buffer) and got good video. Definitely this 1:10 dilution is an easier way to view things, I think
Steven J. Koch 01:50, 18 July 2008 (UTC): I screwed up this sample, but it was tough. Basically, the chip slid around in all directions upon putting the coverglass on, so DNA would have experienced lateral forces in all directions. The sample was very cool to look at, but we didn't see anything striking, if I remember correctly. I think for trying this, the photoresist should be spun on a coverglass if possible
Steven J. Koch 02:32, 18 July 2008 (UTC):The first chip I dropped and it shattered. Nothing really learned, except that if you don't have a little water to form a bond, the chip easily slides all over the place.
The second was very promising! The other guys will be able to explain better, I think, since I had to leave right after we think we found elongated DNA in nanochannels. I thought it looked like DNA, because of fairly uniform lengths of glowing things. However, there's not much explanation of why it would have been stuck like this. We have long movies of what possibly is elongated DNA. I think definitely, combined with above results, is enough great data to make it very necessary to have Deying spend some time making us the exact sample we want for our microscope.
What does it all mean?
Steven J. Koch 02:36, 18 July 2008 (UTC):People were routinely asking me what this means we can do, and I was routinely shrugging it off. I'm still doing that. But I think some main things are:
- We now know that we can easily visualize long DNA molecules
- We have a pretty reliable way of tethering the long DNA molecules. It's reasonable now to pursue other methods if / when we need them. For example, with double- or single-hairpins (digified). We know exactly what we're looking for.
- We have some great videos for talks and posters
- We have the best microscopy I have seen for the nanochannels work. It will be very appealing and be a big spur for projects in that area. What I want to write into my grant is the possibility of analyzing chromatin molecules (nucleosomes labeled with something fluorescent or gold particles). There is also great potential for analyzing features of genomic DNA.
Great work everyone yesterday and today! That's pretty sweet how we were able to have success in such a short time, start to finish.
Steven J. Koch 09:14, 18 July 2008 (UTC):As for the circles, I looked at them again just now: (C:\Movies from Live Feed\png\herskowitz.larryThu17Jul08-17-32-50 guitar strings) and it struck me that these look similar to something I used to see at Sandia when doing microtubule stuff. In particular, when the assay wasn't working well, and MTs were depolymerizing, I seem to remember seeing this kind of stuff. Another postdoc declared these to be "depolymerized tubulin" and I stupidly believed him before I realized that all of his theories were proved beyond a doubt in his mind without ever doing or conceiving of control experiments. Anyway, the only things in common with these assays are: antifade components; dirty coverglass and I think that's all. In those Sandia experiments, there were no DNA molecules, and the fluorophore was rhodamine labeled tubulin. Huh. (I also may be remembering something different.)