"There are two kinds of stuck: not knowing what's going wrong, but having some theories to try and not knowing what's wrong and having NO IDEA why that is or what to do next."
What's the problem?
Problem #1. My gels don't make much sense
Example of what a gel looks like and what it means
One can compare the other bands in the gel, representing DNA of unknown size, to the ladder to tell how big they are. I'm looking for a splice product that is 50 bp long.
Gel Woes ... Ack!
Problem #2. My back-up plan, sequencing, isn't working
Sequencing is the way that I find out whether or not the bands are telling me the truth - whether I REALLY do have my splice product or not. It's failing too.
Once I run the DNA on a gel, I can cut out the band that I think is my splice product with a razor blade (I know - very scientific sounding, isn't it?). Then, I can extract the DNA from it and send it to GENEWIZ for sequencing. GENEWIZ will email me back and tell me exactly what my DNA sequence is that I sent them. That will tell me for sure if I have the DNA I think I have.
What is REALLY Happening
However, the DNA I am extracting from the gel slice is so small, I almost can't send it for sequencing. It also is impure or something. I see no DNA peak in the UV-Vis spectrum on the Nanodrop - which is to say - the DNA doesn't give off its usual signature that pure DNA usually gives off. It looks like ... nothing ... or water or something. It's weird.
At first, I thought that maybe it was because I didn't have enough to detect, but that's not it. Kevin gave me one of his DNA samples that was of an equally low concentration as mine and HE had a nice looking DNA peak. It doesn't make sense.
GENEWIZ Says It's Poor Quality DNA ... hmmm ... purity, purity
Yup. That's what they said when I got my null sequences back. Since I have no DNA peak, I don't blame them.
I tried several times to use the QIAEX II gel extraction kit from QIAGEN, doing a trick or two with it. It hasn't worked. It could be because the silica gel particles used to bind the DNA do not release short sequences well. I read that online. Maybe that's why my yield is low. It doesn't explain the purity though.
I tried to do two other purification schemes on my DNA - using a spin column with agarose matrix to purify, as well as a PCR cleanup kit, for the heck of it. Those didn't work either.
What I'm going to attempt - my last idea
SO. *drums fingers* My last idea to try - the one idea I had left - was to use this method I found for extracting the DNA from polyacrylamide gels that does not involve the kit. Maybe it'll give me higher yield, which will magically solve the purity issue because I'll have more DNA ...? One can only hope.
Asking other people
So far I've asked Sam in biochemistry - an old orgo classmate of mine, now 3rd year biochem pH.D. student - and Becky in the Conticello lab. Neither have many ideas, though both did give me some good guidelines and questions to ask to think about the problem. Khalid said to ask Yue. I shall.
Meeting with Khalid, Victor, Mingda
Spinning my Wheels - Wheeeeeeee!
Meanwhile, maybe I can find more people to track down to ask questions. Maybe. I have my work cut out for me next week. I do NOT relish repeating experiments and doing old stuff over and over. But, it's gotta be done, so it will be.
I know. SO EXCITING, right?
That's research for you. Research is really exciting, except when it's not. Despite that, even though it's so annoying sometimes, I'd much rather be annoyed by perplexing problems than work at something totally uninteresting and unchangeable. We're near the front of knowledge -- who said it would be easy?
My problem isn't that grand. But, it's something like that. You know what I mean. Little annoyances and wrinkles are part of the game. And "the game," as Sherlock Holmes would say, "is afoot."