I dream of pipetting

If you read through the protocol for my mutagenesis, you would notice a lot of "add __ microliters of ___ to ___." We work in microliters (uL) in our lab. 1 US teaspoon = 4,928.92159 microliters. Many of the volumes that I work with are any where from 0.5 uL to 1,000 uL. For those volumes we use special micropipettes (Pictured) 
1 uL would only fill the very tipy tip of this pipette. It is so small, sometimes it's hard to see if you even sucked any liquid up. It's crazy to think that one speck of liquid can change my ENTIRE results. It's scary to know that if I forget one pipetting step, or add too much or too little, then my product will most likely be ruined. Most of the liquid I use are completely clear, so for all I know, I could be pipetting water all day long. Everytime I add a new volume to something, I have to use a new plastic tip on the micropipette. After a rough calculation, I figure I have used over 1,000 disposable tips for the 25 mutations I have created so far. I have 65 more mutations to make. Needless to say, a large amount of my time in lab is spent pipetting, and I have recently started to dream about pipetting.  


I told you I would give an update about the mutagenesis once I was further along. Well, as I said in my earlier post, I chose 15 residues to mutate on the protein that makes up the transporter. Each of those residues gets 2 different mutations. One mutation will change the space it takes up, and the other mutation will change the charge that it has. Each of these mutations needs to be inserted into 3 different DNA templates, for comparison purposes. That means I have 90 mutation. So far I have created primers for all of them, but I have only started the mutagenesis on 25. 


The DNA template for the serotonin transporter is somewhere around 4,000 base pairs. I only need to change 2 or 3 base pairs to make my chosen mutations, so we ordered just the tiny segment of the DNA with that mutation in it, called a primer. In order to get the mutation into the template, I ran a PCR (polymerase chain reaction), which is used to amplify a small amount of DNA into a large amount of DNA. Inside a reaction tube I added the un-mutated template, the forward primer, and the reverse primer. During the cycle in the PCR, the double stranded DNA template is pulled apart, and the primers will stick to the template in the place where we want the mutation. The cycle will continue and replicate the DNA with this primer attached, creating a mutation. Once the cycle is over we are left with 20% un-mutated template and 80% mutated template. I then digested the product in an enzyme that cuts up the un-mutated DNA. After that, I transform the DNA into E. coli cells. 

E. Coli is a bacteria that loves to pick DNA up from the environment and start replicating it with it's own DNA. By using bacteria, we can make LOTS of DNA because the cells replicate really fast. I grew the cells up on plates of agar. Once colonies started growing, I moved the colony into a broth for it to grow even more. Once I grew enough E. Coli, I was able to do a miniprep. A miniprep is a procedure used to purify the DNA out of the cells. Basically, we burst the cells, suspend the mutated DNA that we wanted, and purify it out from the chromosomal DNA of the bacteria. At this point, we have to check and make sure we created the correct mutation, and didn't make any mistakes along the way. We use  gel electrophoresis to check, and once we think we have the correct mutation, we send it away for sequencing just to double check. It's a really long process that I didn't think I was going to have to go through. I wish we could just order the DNA that we want and be done with it.....haha.


Hopefully the mutagenesis won't take too much longer....I really want to get started on the actual experiment.