I Do Science II - The Wrath of Kits

September 2, 2011

Rheanna Sand

And now, the continuation of "Wait! I Do Science!"... a live blog that allows Rheanna to update her website AND finish her PhD. simultaneoulsy!


Hey everyone! You've probably been checking this site every 5 minutes waiting...nay, see some more pictures of my riveting experiments. Well, your wait is finally over! Here is my progress over the last week, in all it's molecular glory.


When I left you last, I had re-done what we call a "digest," which is just using special proteins to sever the DNA in a very particular place so that the ends become "sticky." So, what I expected to see were bands, or glowing lines, that match up with the ladder at about 600 base pairs (bp). The lane on the far left is the ladder. The brighter bands correspond to certain sizes - the bright one furthest down is 500bp, so I wanted to see bands just higher than that bright band.



Woo hoo! They are there! But not quite bright enough so that I can remove them from the gel and still have enough to work with. After redoing it with more DNA and cutting these out of the gel, I used a "kit" called a gel purification kit to purify the DNA. Only then could they be "glued" together by another protein called ligase. In the end, I basically I took the bright band in the rightmost lane and glued it to each of the 600bp bands. That happened last Friday. I didn't take any pictures because, well, it's just a tube with liquid in it.


But how do I know the gluing process happened correctly? I won't know until I take my shiny new mutant pieces of DNA and grow them in little DNA factories - E. coli! Mmmmmm, smells like hoarding!


We call this process "transformation," since the bacteria are taking up the DNA as their own and therefore becoming a different genetic entity (but I also like to believe the E. coli have a spiritual awakening and truly "find themselves" when we electroshock them). The transformation itself is very quick - BEEP - but the recovery takes an hour or so. Then I spread the garbage-smelling mixture on an agar plate that contains an antibacterial agent. That way, all the individuals that didn't take in my DNA present die. Take that for being ungrateful! The ones left over grow into colonies of thousands of individuals, which appear as a pinpoint on the plate the next day.


I then take one of these dots and scrape it into a tube with nutrients and put it at E. coli's favorite temperature - 37 degrees Celsius - overnight in a rotating shaker. If the mixture goes from clear to cloudy, I know it's full of bacteria (which explains the slight feeling of revulsion I get when I drink a cloudy beer).



From here I use another kit to purify the DNA that I originally put in. The difference is now there are thousands upon thousands of copies! Thanks E. coli!


But how do I know that I have thousands of copies of the RIGHT thing? The fact that it grows in the antibiotic doesn't mean the DNA sequence is exactly what I want. It could be the original, non-mutant DNA. I must do a sequencing reaction to find out. This is nothing short of a modern miracle, and I always wait with baited breath to get the results.


Which I will share later on today... but for now, I must get back to work!


Update (5:30pm MST): The sequencing was a success! You can tell by the perfectness of the chromatogram peaks in the image below, each signaling one of the four bases in DNA, adenine, guanine, cytosine, and thymine.


As for whether my "playing God" worked or not... well, let's compare my sequences with the sequence it SHOULD be:


The first sequence is the original. The second one is what it should be changed to. The A1, A2, and A3 are the sequences I created. They all match! Success! In the end, all but one of my sequences was correct. This means that I have "good clones" for all three mutants I was trying to make. When I started, I was trying to make five... so I did lose some mutants along the way. But I can do a lot with these three. That is, if I can make good mRNA...


To be continued!



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