What happened exactly? Well, I ran another 20% gel of my 10 and 20mer oligos (10 and 20 nucleotides). I was being a bit too optimistic. I made lanes in which I loaded 50 nanograms (what I usually would do) but also lanes with 10 and 1 ng and one with 200 pgrams (picograms). My *thought* was that I would find the limit of sensitivity with these lanes - but that was a bad idea, I think. I need to load them all the same. I had a bad feeling about it.
The good news is the fluorescence worked. The bad news is what I got was terrible. It was EPIC in its total badness. Very embarrassing. I suddenly realized how many stupid things I had done and how much I needed to fix. I wanted to headdesk myself on the terminal and knock myself out. But it's ok.
Way too much background fluorescence. I need to rinse off the gel to avoid that. (*lightbulb* Doh! What a good idea. Read it in the typhoon manual.) I also need to read the Typhoon manual - it might give me some useful insights on fluorescence. I need to load all lanes with one DNA concentration. I'll stick with 50 ng. They were the only bands that showed up.
I'm not yet completely sure why my sensitivity is so low. Yes - my DNAs are small - that could be the issue. But, the papers I'm reading have such nice gel pictures and I'm left wondering ... how did they DO that??? For example, the paper by Raines that Kevin and Ian used as their primary does a ligation reaction with 200 pmoles of DNA and 40 pmoles ... how am I supposed to detect DNA at those concentrations, I wonder? And why don't they give any specifics AT ALL, even in their supplemental? I need to check out a few more papers I've collected and see what I can find. Answers are there somewhere.
The dye I'm using, SYBR gold, is supposed to be able to detect 25 picograms of DNA. The question is - at how many base pairs? I think I read it detects dinucleotides. I need to look this up again too.
However, the real question is how to get this protein to dissolve. I'm postponing any more oligo imaging until I can get that solved. I just wanted to do at least one fluorescence image to see what would happen.
I made a bunch of different solutions of it today varying salt and DTT. I found out that the way I was doing it wasn't really that good of a way. It was annoying. I had to measure out tiny amounts of salt with grains greatly varying in size. Add one grain. Change the weight by 10-times too much. Grrrrr.
Meanwhile, Kevin made a comment about a movie in which Brad Pitt went to save his love from some Asian jail - which started a vibrant discussion about which one it was. I was alternating laughing with saying, "good grief" and huffing (giving my salt the evil eye). It was funny. Kevin seems to tend to say completely random things at times, which is terribly amusing.
To Conclude - Random Comments
I did make 10x TBE finally! I found out that to make up a solution of EDTA first is not a good idea per se - at least, if you do, you don't want to pH it. The Tris-base does that for you after you add it. Then, adding the boric acid, and ta-dah! Buffer is made. I feel like I understand that buffer way better than I did. That thing was irritating to figure out how to do.
I now have it on my bench labeled as "Jessica's special 10x TBE." Whatever that means. My thought was the lab might not want to use mine, in case I did it wrong. I think I'll make more to fill the regular bottle of it too now that I'm sure it's good.
I didn't get done what I had planned. Ran out of time - but that's pretty typical. I usually over plan. But rather over plan than under plan.
Dr. Salaita called this morning at 10 a.m., which we think is something like 2 or 3 a.m. in Jordan.
James is playing EN 2 - the game with a little stick figure man that runs around collecting gold coins and trying to get to a door at the end of a maze, but usually blows up on mines instead.
James said he read something that said the majority of time people spend playing games is spent failing and trying again. I felt there was a parable on life there. That's the majority of time people spend in science as well. But then again - there is only one way to succeed and many ways to fail - so of course, one will fail most of the time because you can only succeed once. Unless there are many ways to succeed. But still, once you succeed once, you don't usually look for another alternate method to do the same thing - unless it's a rare case.
Also, this morning, I was on Scenic Hwy. The light turned green ... and a mother goose and her flock of chicks picked THAT MOMENT to walk across the street. It was hilarious!! I took a picture. The cars behind me were SO mad and honked like crap at me. But what could I do? I laughed.
Last Weekend (a boring summary)
On Saturday, John played in a qualifying tournament for Warmachine/Hordes. I hung out and watched for an hour, ate a sandwhich and took a nap in the car for an hour and then wrote up experiments and read about buffers for the rest of the night until 10 p.m. I got home at 1 a.m. exhausted.
On Sunday, John and I went to Bryan's house and he did his role-playing session with Bryan, Matt, Steven and Walter. I listened, made brownies, took a two hour nap and wrote up a schedule. I also played a round of Citizens of the Multiverse at the end. I went to bed at 10:30 p.m. exhausted.