Even more PCR and Electrophoresis (Legionella pneumophila Blog Week 7)

Hello everyone, this is week 7 of our journey through the S-STEM internship!  This week for our Legionella p. project, we had to run PCR on 10 different samples.  We ran the PCR on Tuesday and today we ran the amplified DNA in the Agarose Gel. Unfortunately, I goofed on 4 samples that I loaded (I loaded the gel wells with 7 microliters [μL] of material instead of 12μL).  Our mentor, Robin Cotter, advised us that it would be wise to run the PCR again on these samples and do the electrophoresis again in order to verify our results.  The reasoning is that; if the Legionella p. bacteria are present but there was not enough genetic material in the 7μL then it may not be visible when looking at the gels.  As for the remaining 6 samples that did run and ran well, we will be cutting out the clearly visible bands and performing a DNA extraction process from the gels so that we can send the samples to ASU for genomic testing. Once we know the sequence of the nucleotides, we can compare that to the known sequence of Legionella p. and know for sure that the bacteria were present in the samples that we collected.

That is all for now! Above is a picture of me on my birthday with Daisy one of my group mates.

P.S. Today is my birthday!

1st Agarose Gel Pour/Run (Legionella pneumophila Blog Week 6)

Spring Break, short are thee
In your midst I wish
to be.  Once more free
from the petri dish

Yet here I am at,
close to the midnight.
Had I not laid flat
I'd be far from plight.

Hello everyone, that was my sadness from the end of spring break coming through.  I'll jump right into it since there is no better way to do work.  This week in the Lab our team ran PCR on nine samples some of which had been collected nearly 6 months ago.  I'm sure many of you are familiar with this process but after the PCR had been run. We poured 3 gels and ran our samples through 2 of them.  This was the first time I had ever poured the Agarose Gel and it being my first time it did not go well at all.  Luckily Daisy poured the other two and those were the ones we were actually able to use.  Below is an image of the water samples collected.  According to the banding size it appears as though we actually got a positive for the Legionella bacteria, on the first go around!  We didn't have enough time to scan the second gel but once I get the picture I will upload it onto here.  Coming back hasn't been so bad after all!
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NanoDrop Pioneers! (Legionella pneumophila Blog Week 4)

Last Friday Josh showed our group (Daisy, Zaira and me) how to use the new NanoDrop machine! It was awesome to be the first ones to learn how to use it and actually get to run our samples through it.  Daisy even taught Bethany how to run her samples in the NanoDrop. 
     The NanoDrop measures the concentration of DNA in the any given sample.  It also has the ability to determine the purity of DNA in a tested sample by measuring the ratio of absorbance measured at wavelengths of 260nm and 280nm.  DNA tends to absorb at a wavelength of 260nm while protein, phenol and other contaminants absorb at a wavelength of 280nm (NanoDrop, 2007).  By comparing the two measurements, the NanoDrop is able to determine purity of DNA.  A ratio measurement for reasonably pure DNA would be around 1.8.  As you will see in the attached image, our samples do not display this type of purity.  The lowered purity is expected because our samples, as of now, do not contain purely DNA. The samples have other contaminants that affect this reading.  Daisy and Zaira have cut DNA samples out of agarose gels after PCR in the past and their results actually were aligned with what would be expected for pure DNA.  So we will get there soon!

NanoDrop, http://www.bio.davidson.edu/gcat/protocols/NanoDrop_tip.pdf

Doing Some Actual Work (Legionella pneumophila Blog Week 3)

I finally had the chance to do some actual work this week involving the collection of samples and working on extracting genetic material from the sample collected.  Oliver and I collected a sample of water from the cooling towers and we will be filtrating and extracting (if there is any) genetic material from our samples this week.  

Up until now I had been doing a great deal of background reading to familiarize myself with the bacteria, its significance and the methods that we would use in order to positively identify the presence of Legionella pneumophila.  

First up in the process was the actual collection of the sample.  Down below you'll see the location and a brief description of where the sample was taken from.  Once the sample was collected, it must undergo a filtration process in order to concentrate the contents that were in the sample into 5mL of water so that we can work with it better in the lab.  (FYI: Video is loud)

Once the samples have been concentrated the DNA (if there is any) of whatever was in that water is extracted from that sample using a Soil DNA Extraction Kit.  What this kit does is, take all of the genetic material that was in that sample and extract it from the organisms that were residing in the sample of water.  

This was my first time going through all of these steps and I think I was able to get through everything successfully, with the help of my teammates (Daisy and Zaira) of course!  I guess that's all for now, tomorrow we'll get a chance to use and learn how to use the new NanoDrop machine so I'm really excited about that.