Research Results: Enzyme is Active!
Now: Where We Go From Here
Discovery 1: My old enzyme was not bad - it was the RNA
I ordered new RNA and isolated new enzyme. I tested the new enzyme on the new and old RNA strands and my old enzyme on the new and old RNA strands. What I found was that both enzymes worked great on the new RNA strands and poorly or not at all on my old strands.
Conclusion: The 3'-phosphate on my RNA had hydrolyzed off. Dr. Weinert had told me that this was likely and happened to her cyclic phosphates within a month.
Nerd Note: For the curious, 5' and 3' refer to positions on the ribose sugar of each nucleotide in DNA - it is the location at which each base can attach to another base and extend the DNA chain. A DNA nucleotide is made up of a nucleobase (A, C, G, T), a sugar and a phosphate.
Discovery 2: My enzyme is not active in the conditions I used for my splice reactions
A Bet: Khalid and I Have Challenged Each Other
I think the experiment showing this fact was very clear, but I did scale the concentrations down, using the same ratios. Khalid thinks if I scaled them back up to what I actually had, with the same ratios, the added concentration itself might solve the problem. I insist he's wrong. He's sure he's right, because of enzyme kinetics. My hunch is based on a gut instinct about my enzyme - so I admit - he has the upper hand.
Therefore, Khalid and I have bet lunch on this point. The loser buys the other lunch. We will SEE!! I'm so convinced I'm going to be right about this. Only next week will tell.
Discovery 3: DNAzymes can inhibit rtcB activity at high enough concentrations
Where do we go from here? The next questions:
1. What are rtcB's working conditions?
2. Do DNA splints hinder the splice reaction?
3. What is the activity of my DNAzymes?
FIN: Going Forward in Future Weeks - 2nd Year Report
This is part 1 of the pass/fail for the pH.D. Part 2 is the oral presentation, which I hope to schedule for early October. I *will* still be doing probably one experiment a week, to keep me from going stir crazy.
THUS, IT MUST BE!
I've also signed up for two classes: 1) Biotechnology in Chemistry by Dr. Lutz and 2) Physical Organic Chemistry by Dr. Ahmed. We will have an ethics class and seminar/grant writing class as well. I can't wait until after all this is over, so I can go back to experimenting. It's so much fun!