User:Anthony Salvagno/Notebook/Research/2009/11/18/Ligation Results
I accidentally let the gel run for too long, so I filled a pipette tip box with my cybersafe TAE buffer and am now letting it soak for about 30-45 min. The reason I let the gel run for so long was that after updating Brian's notebook I decided to check my not so accurate numbers just as a comparison with what Brian will get. I got so caught up in the data analysis that lost track of time. I will make a new spreadsheet of this information and post it later in another notebook entry.
- DNA Ladder 1kb
- anchor-BstXI/adapter/EarI-pBR322 fragment unzipping construct
- anchor-BstXI/adapter/BstXI-anchor diagnostic construct
- anchor-BstXI/adapter/BstXI-anchor unzipping construct
- Note: That thing on top is some reflection from the camera, cause it wasn't in the gel.
Fucking awesome, this is the sexiest most beautiful fucking amazing thing I have ever seen in my life. I want to take my ligations on a smoking hot date tonight to the best restaurant in town, tell them how much I love them, and then make the best sweetest passionate love to it, and then fuck it's brains out. That is how stoked I am to see this. I have definitive successful ligation of the unzipping construct.
Let me repeat that...
I HAVE DEFINITIVE SUCCESSFUL LIGATION OF THE UNZIPPING CONSTRUCT!!!!!!!!!!!!!!!!!!!!!!!!!!
Let me explain in long gorey detail my thought process throughout this whole ordeal:
- 3 years ago, Koch explains to Larry and I the foundations of the project. We are elated and join the lab.
- More than 1 year ago, Koch tells me he thinks I can lead the lab in Molecular Biology and would be really good at it. I agree.
- 9 months ago, I go off to Osley lab to learn all about Molecular biology and to do unzipping construct creation.
- 6 months ago, I leave Osley lab with a small percentage of tetherable DNA.
- Around that same time I come back to Kochlab and try to pick up where I left off.
- 3 months ago, I restart unzipping construct creation to try and perfect the method so that I can get definitive Shotgun DNA clones.
- Days go by and all I have are failures. Koch releases deadline to get something unzipped by the end of September. I fail.
- With each failure my confidence in my abilities shrink. Koch begins to think that maybe I am not so good at this as he believed. I have faith, but no proof. Every ligation failed. How could every ligation fail? There are too many variables and not enough time. I have to figure out how to determine what the problem is. Is it the anchor? Is it the digestion? Is it the adapter?
- My A-exam approaches and I just want to see successful ligation. That is all I want. I work super hard to get the tweezers active again. No ligation success.
- Koch and I reset. We discuss possible problems and how to troubleshoot. We get all new oligos to make sure it isn't the adapter.
Fast forward to today. I wrote up some stuff earlier in the week to do ligations yesterday. Time ran out so I had to analyze the results this morning. I had already figured that the reaction was a failure before I ran the gel and was thinking of how to adjust. I run the gel. I run the gel for too long. I decided to do a quick analysis before I allow my gel to sit in CyberSafe to restain. On quick analysis it looked like I could see 1 band before the stain disappears. I thought this band is a bit high for a failure, but I must be wrong. Let me stain and see officially. After staining, Larry and I go out for lunch. When I return I analyze the gel. I look and see some peculiar sight. "What is this? 3 bands? Those other 2 lanes only have 2 bands. Hmm, what is that? And why are those two bands lower than the middle band in lane 2?"
That was what I thought, and then I had an epiphany and yelled out in excitement as I realized I had achieved success. That high band is ligated product in all it's splendor. Those other bands are double the length of the bottom band. Why? Because I am ligating anchor to anchor! It worked too!!!! OMG, I can't be any happier right now! I feel like Nate in the 2006 NBA Slam Dunk Competition. In the second round, Nate had missed like 15 straight dunks and finally gets one. When the ball bounces back down he slams his fist into it as if thanking the basketball gods for letting him complete the dunk. Check it out at 1:29 in the video:
<a href="http://vids.myspace.com/index.cfm?fuseaction=vids.individual&videoid=1116392" style="font: Verdana">2006 slam dunk contest</a>
<object width="425px" height="360px" ><param name="allowFullScreen" value="true"/><param name="wmode" value="transparent"/><param name="movie" value="http://mediaservices.myspace.com/services/media/embed.aspx/m=1116392,t=1,mt=video"/><embed src="http://mediaservices.myspace.com/services/media/embed.aspx/m=1116392,t=1,mt=video" width="425" height="360" allowFullScreen="true" type="application/x-shockwave-flash" wmode="transparent"></embed></object>
<a href="http://profile.myspace.com/index.cfm?fuseaction=user.viewprofile&friendid=23165171" style="font: Verdana">Keanu</a> | <a href="http://vids.myspace.com " style="font: Verdana">MySpace Video</a></html>
I just punched my eppis in the ground thanking the biology gods. Finally...