Spring 2016 Running the Gel

This week in lab I took the DNA I extracted last week and ran a gel electrophoresis gel with it. The protocol I followed was used to extract DNA from black widow webs, and I wanted to be sure that the extraction process worked on other types of spider webs as well, so I ran this gel to prove that I actually had extracted DNA from a variety of webs. This is what the gel looked like:

 And here is the same gel with molecular weights and lane detection added in by the computer the fancy new gel imaging system can do:

So the thing is I put DNA in all but the last three wells but only two lanes are showing up on the lane detector. This means that I probably don't have DNA in those wells and the reasons for this are: not enough supernatant was moved from the extraction tube, or I left the DNA at room temperature too long and the DNA degraded, or I didn't use DNase free micro-centrifuge tubes and the DNA degraded, or I left the gels in the light too long and the syber green dye degraded, or I pipetted the DNA and loading dye wrong and didn't get any in the actual wells, or any number of other things. There doesn't seem to be a strong correlation between the type of spider the web was collected from, or the age of the web, and whether or not DNA was detected. In lane four, or two on the lane detection image, there is a strong clear band at 120bp. This was a web that was collected almost as it was being spun, as the spider was still in the web and construction was not completed yet, which may explain why the band is so clear.

Spring 2016 First DNA Extraction

This week in lab I performed my first spider web DNA extraction and my second extraction ever!

Whenever you try something new you learn a few things. While doing extractions this week I learned: I should make sure my microcentrifuge tubes are free of DNA-degrading enzymes called DNases, there's a big difference between 1.5 mL and 2.0 mL when working with the tiny volumes used in DNA extractions, and that to keep DNases from being activated, the DNA must be on ice nearly constantly.

I am unsure if my extraction went well enough for me to prove I have DNA from webs just yet but I'll get it soon!

Spring 2016 Week 3

Another awesome week in the lab.

This week I diluted ammonium acetate to be used in as a protein precipitating solution. All DNA is surrounded by a vast array of proteins. There are proteins in the cell walls and cytoplasm as well as chromatin proteins actually in the strands of DNA itself, wrapping the DNA into tightly wound bundles. The proteins can contaminate DNA samples, making tests hard to decipher. So to get rid of proteins and other cellular junk ammonium acetate is added to the solution. This caused the proteins to solidify out of solution, leaving the DNA behind.

This week I also collected some spider web samples to test my extraction process out on. I don't want to use up sources of black widow DNA just to make sure I can get DNA from webs, so I went out and collected samples from other species I could find on campus. I collected cellar spider webs from inside the lab, some abandoned looking web that could possibly have been a widow's web and the web from an orb weaver spider that is found on the chemistry department building. The orb webs were a little yellow in color, which I thought was super cool. The possible widow web might have too many leaves in it to use, but all the samples I collected from the environment had at least one contaminate it it: human hair, cat hair, leaves, dandelion seeds etc.  Corri says that most of the contaminates will be washed out while the DNA is being processed, and because the primers we're building should be specific to spider DNA, only spider DNA should be amplified during PCR. We'll see.

Here is a picture I took of an extremely patient spider whose web I collected.

Spring 2016 Week 2

This week in lab I began to gather and prepare the chemicals I will use for my DNA extraction. 

To remove DNA from a eukaryotic sample, the first step is cracking open the cell's various membranes to get to the nucleus, where the DNA is stored. Lysis buffers are solutions of chemicals that break down the cellular membranes of a sample and also cause proteins to precipitate out of solution. Lysis buffers are generally alkaline and contain detergents, chemicals with both hydrophobic and hydrophilic portions of the molecule. These detergents and the alkalinity of the solutions cause the cellular membranes to degrade by disrupting the hydrophobic membranes. Other chemicals can be added to the lysis buffer to regulate pH and to cause unwanted macro-molecules, such as proteins, to precipitate. 

So this week in lab I made my lysis buffer. None of the commercially available buffers had the same concentrations of chemicals as the protocol I'm following. I had to make 10mM Tris, 10mM EDTA, 2% SDS pH 8, from more concentrated chemicals found in the lab. It was great to practice dilution again. Diluting chemicals is a precise method that I feel is not one of my strengths and thus something I must practice more.