1st Milestone achieved! Splice product found!!
Story of the marathon experiment!
On the infamous Thursday, I suddenly got my primers that I had ordered and my RT-PCR kit came in the day before. I had an experiment planned already - trying to figure out why my DNase I was not digesting my DNAzymes. But I looked at my supplies and was like ... I'M JUST GOING TO GO FOR IT - YEAH!!!
Insanity starts - GO GO GO!!!
So, I started a marathon experiment dash! I'd never done RT-PCR before or PCR by myself (did it as an undergrad in the Kushner lab). I began like mad, starting reactions and more reactions! I've never done so many at once before. I was reading the manuals on the go, literally never sitting down from morning until evening except to write in my lab notebook.
I had three DNase I digests going to test whether or not it would work without salt (my original experiment). Then I started a DNAzyme digest of my RNA substrate for 1 hr. After it was done, I promptly started an rtcB ligation reaction with and without splint. After the ligations had gone for 3 hours, I started one hour RT-PCR reactions on them, then doing six PCR reactions, getting help from the Lutz lab on their PCR instrument. I ran a gel on the DNase I digest and also on all the PCR reactions and DNAzyme digest.
In all, that was 3 DNase I digests, 3 RT-PCR reactions, 6 PCR reactions, 2 DNAzyme digests and 2 rtcB ligations and 2 gels ... PURE insanity!
Me in lab late
This past week: confirming the results and solving problems
This past week, I did another ligation and PCR to confirm the results of the crazy experiment. It looks hopeful! There is some ambiguity that I'd like to erase by sequencing the gel products. I have to look into that in this next week and figure out how to do it. I already got my account setup with Genewiz for sequencing, and training with Emory Express so I can send orders.
Problem Solving: The Next Step in the Saga - dye attachment!
I'm having two major problems right now.
Problem #1 My RNA I'm transcribing is not staining well at all, unlike my last transcript design, and I have no idea why. I have to use insane amounts of it to see it at all.
Problem #2 DNase I, a commercial enzyme for cleaving DNA, refuses to cleave my DNAzymes before my ligation reaction and their bands are obscuring others I need to see in my gels. Even though it worked without salt, when I try it in a real reaction - poof! doesn't work again!
To solve these two problems, my plan is to attach a fluorescein - a fluorescent molecule - to the 5' end of my RNA. This will allow me to see just the bands I need to without worrying about the DNAzymes (which have no dye attached), while also helping my RNA be visible - killing two birds with one stone.
How will this be done? Methods of dyeing RNA
There are several ways of attaching dyes to RNA. The easiest way is to purchase an RNA with a fluorophore already attached. However, my RNA is far too long to buy (insanely expensive), which is why I'm transcribing it. Therefore, I needed a way to do this manually on my homemade RNA sample.
One can incorporate fluorescent bases into the RNA strand as it is being transcribed, or modified bases that had attachment points for dyes with EDC chemistry later. However, we worried that the modified bases would hinder DNAzyme cleavage.
The method I chose: 5' modification
It turns out, there's a way to label the 5' end of an RNA by reacting ATP-gamma-S with T4 polynucleotide kinase (T4 PNK) and the RNA. It will transfer the sulfur-modified gamma phosphate to the 5' end of a de-phosphorylated RNA strand. A dye with a reactive group can then be used to react with the sulfur and attach to the 5' end.
Picture of ATP-gamma-S and a regular ATP
I therefore ordered some of this modified ATP and transcribed new RNA. After I had dephosphorylated it with shrimp alkaline phosphatase, I purified it on a column. I got my ATP in the DAY AFTER I ordered it!! And I finally got the fluorescein dye I'm going to attach in this past Friday.
Other experiments in the past week: EPIC FAIL!
My primary objective is to label the RNA, then do a DNAzyme digest of it and a ligation and see how things stand. I'm hoping this labeling will work and clear up a lot of the confusing bands.
I also discovered an error with my splints that I'm using to splint the ligation reaction. I ordered another set for the experiments next week. Maybe I'll see more ligation product with a correct splint!!
My secondary objective is to figure out how to sequence the bands in my gel.
Lastly, I have a research talk next Friday ... SO ...