Wednesday, June 18, 2014

Tyrannosaurus Rex

I am sorry I have not had time to write a blog post recently.  I have been REALLY busy.  Yesterday, I finished my third box of tips!  There are three different tip sizes depending on the amount of liquid you need; I have now finished one of each size.  

Recently, I have been working on recombining my mutant DNA into a plasmid that contains GFP.  GFP stands for green fluorescent protein; it is a tag that you put on one side of the DNA so that you can track it in the cell.

The image above is an outline of what I am trying to accomplish next in lab.  I have already amplified my H3 gene, and completed the reaction that places it into the pENTR vector.  The next step is to create mutant DNA.  This process is called mutagenesis, and is accomplished by using primers that contain the mutant DNA sequence, and is able to bind to the wild-type DNA.  The primer is able to bind to the wild-type DNA due to a complementary sequence.  The DNA sequence contains four base pairs: A, T, C, and G.  Base pair A will bind to T, and C will bind to G.  After the primer is created, using the original sequence and the mutant bases, it binds to the wild-type DNA, and an enzyme will copy the rest of the sequence (the sequence not contained within the primer).  This process creates a plasmid that contains the mutant DNA.  I have created one of my mutant sequences, and I am waiting for my other mutant to be sequenced (this will allow me to determine that the mutation was properly incorporated into the DNA).  Next, I want to recombine the mutant DNA into a plasmid containing GFP.  The red mark is the GFP tag, and the green mark contains DNA that is called the "death gene".  This means that if the mutant DNA is not inserted into the plasmid in place of the green gene, the cell with the green gene will die.  I am currently having problems with this step, and I am unsure why.  To determine if the mutant DNA is inserted, we run a gel (figure explanation below).  My two outcomes have been undigested DNA and DNA with no plasmid insert, meaning it contained the death gene and should have died.  I am currently trying to redo this experiment.

The laboratory I work in houses two different professors, and although we have been working side by side for two weeks, we have not officially met.  Thankfully, that changed last night when we had lab bonding.  We hiked a small part of Mt. Baldy to a river.  Currently, there is a drought in southern California and the river was nearly dry.  It was a great way to get to know each other.

Living on my own has been a great experience, and has taught me to become more independent, especially when it comes to food.  Today, I had a lot of great foods.  This morning, I decided to make chocolate pudding.  I decided to experiment a little, and added 2.5 instead of 2 cups of milk.  The taste was so rich; I cherished it for most of the afternoon, eating small bites as I wrote another paragraph of my Thesis.  In addition, I went to My Delight Cupcakery and ate a Chocolate Caramel Cookie Crunch Cupcake (that's 5 C's for you! One for each of the colleges).  It contained caramel ice cream in the center of a chocolate cupcake, chocolate mousse, and was edged with Oreo and toffee.  It was heavenly :)  If you have never been and live in the area, I suggest you go IMMEDIATELY!  I have not found another bakery that makes a better, or even equal, cupcake.  They are never dry, and are made to perfection.

However, I still had not eaten dinner and wanted some protein.  The last food item I ate today were the best meatballs in the world: My Dad's!  I promise that you have never had a meatball better than this one.  It is phenomenal!  It is very time consuming to make (sorry Dad), however I believe it is worth all that time solely due to the flavors, texture, and taste. Nom nom nom.  If you thought that the cupcake sounded like the best food item of the day, you may in fact be wrong.  This meatball MIGHT out due the cupcake, but not by a lot!

"A creative man is motivated by the desire to achieve, not by the desire to beat others." --Ayn Rand

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