PCR Time !

 Last week I worked on my extractions. I did about three extractions using the same protocol and the same bacteria. If you have been following up with my experiments you now know that the last extraction was definitely the best I have done and the results from the gel electrophoresis can prove it. Now that I have mastered the extraction part of my experiment running a PCR for each bacteria should be easy.

Let me remind you that PCR stands for Polymerase Chain Reaction, and what this type of machine or technology tool does is it takes a piece of DNA and amplifies it by making several thousands to millions copies of a specific DNA sequence. This makes large and efficient amount of DNA that could be used for several tests. For example finding out suspects from a criminal act, finding out who a deceased body belongs to, or track down virus and diseases. However in my case I am just running this experiment to see how successful I can be.

In order to even begin a PCR I must have the DNA of the bacteria extracted and ready to go, because I used E. Coli, Bacillus, S. Aureus, and Agrobacterium, I continued with the PCR protocol from these same extractions. The PCR works a little different from the gel electrophoresis because for one it takes six times longer! We add different reagents and components such as master mix, primers, DI water and of course DNA, the order in which we mix these four components and reagents together is important and when doing this we must keep them on ice. Primers are reagents that contain nucleic acid and are included in this mixture because this nucleic acid helps start the DNA synthesis. The master mix is a reagent that contains Taq polymerase, and this polymerase helps amplify short DNA segments. The addition of water helps dilute the concentrations of both reagents just mentioned and DNA has to be included. I ran two PCR the first one was with primers 4 and 5 with agrobacterium and E. Coli. The second PCR I tried was with primers 3, and 4 with E. Coli, S. Aureus, Bacillus, and Agrobacterium. To check if the PCR produced DNA I run a gel electrophoresis just like I run it for the extraction part. I was not successful with the first PCR and failed to capture a picture of it, but the second try was a lot better.

 

PCR Steps
1. Add 10 microliters of master mix
2. Add 5 microliters of DI H2O
3. Add 4 microliters of primer
4. Add 1 microliters of DNA Sample



Primer4
E. Coli, S. Aureus, Bacillus, Agrobacterium

Primer 3
E. Coli, S. Aureus, Bacillus, Agrobacterium

Third times the charm




Figure 1
Ruler  E. Coli S Aureus, Bacillus Agrobacterium
 

Last week I worked on my DNA extractions. The bacteria I worked on were E. Coli, S. Aureus, Bacillus, and Agrobacterium. To test if my extractions were successful I ran a gel for each. I have some pictures of what I see after running the gel. I allowed the gel to run for 35 minutes at 120 volts, when that was done I placed it under UV light, and that light allows us to see if DNA is present or not. The reason we check if DNA is present this way is because to each sample I add two different dyes if any of you recall, one of them (orange dye) gives the DNA bonds higher resolution  in the agarose gel while the syber green stains the nucleic acid bonds from the DNA allowing it to absorb blue light (UV light) and emits a green color. When I place the gel under this UV light I should be able to see green light for each of my samples showing the existence of DNA including the ruler. For example in figure 1 (my first extraction), you can only see about three bacteria not including the ruler. The ruler has about 5 bright dots in its row. You can only see a big dot for E. Coli, a small dot for Bacillus, and just a line for Agrobacterium. This extraction worked for those three bacteria but failed for the S. Aureus, but none of the samples actually ran all the way down like the ruler did. This gel is 1% agarose a normal percentage to use for a gel, but in this case this gel was too concentrated and for this reason the samples did not run all the way down like they should have. If you notice the only bright bands you see at the other side of the rectangle is the rulers, not the bacteria's DNA. On figure 2 (my second extraction) though, you can see a bright light for all of them, even though you can barely see the S. Aureus band. This extraction worked for all four but even though the gel concentration was changed to .5%, the samples did not run all the way down. Figure 3 (my third extraction) though is the best extraction I have done so far. You can notice the difference in the brightness, and the length that each travelled ( almost to the ruler). By length I mean how far each sample ran to the bottom part of the rectangle.




Fgure 2
Ruler E. Coli S Aureus, Bacillus Agrobacterium
 

Figure 3
Ruler E Coli S Aureus Agrobacterium Bacillus
 

QuickExtract Bacterial DNA Extraction Kit

Equitment for DNA Extraction

This week for my internship I had the chance to proceed with my DNA extraction experiment. Remember my goal for all of these experiments is to fine an efficient way to extract DNA from bacteria that will create clear PCR product.

Recall that last week I made cultures for four different bacteria, E.Coli, S. Aureus, Bacillus, and Agrobacterium. I continued with the extraction procedure the next day. A summary of my procedures for the DNA extraction are the following:

1.  Extract 500microliters of culture into a flat cap tube.
2. Centrifuged the bacteria at 1300rmp in a micro centrifuge for 3 minutes. (To pellet the cells)
3. Extract the liquid (leaving only the pellet of cells) and add .5ml of sterile water.
4. Recentrifuge at same speed as before for 3 minutes.
5. Remove and discard the supernatant, and add 100 microliters of QuickExtract Bacterial DNA Extraction Solution to cell pellet.
6. Add 1 microliter of Ready-Lyse Lysozyme Solution to each tube and mix.
7.  Incubate at room temperature for 15 minutes. 

When all these steps were done for the four tubes, they were ready for either PCR, or for running a gel. However because both of these steps require time they were incubated at 4 Celsius degrees, and left there over weekend.



Molecular Weight Ruler

On Tuesday, I ran an agarose gel electrophoresis (method used to separate DNA based on its size). Syber Green is a blue/green cyanine dye used to stain nucleic acid bonds to DNA (Absorbs blue light and emits green). Orange loading dye gives bonds higher resolution in the agarose gel. Both dyes are used for this procedure in addition a separate ruler to compare the results of the DNA. The electrical field that is applied to this apparatus is set to 120 volts, the entire box where the agarose and DNA are is completely covered and it runs for 35 minutes. 

Syber Green and Orange Dye

Flat Cap Tubes for Agarose Gel Contain: 

• 8 microliters of DNA
• 2 microliters of Orange Dye            
1 microliters of Syber Green

 Ruler Contains:

• 9 microliters of Molecular Weight ruler
• 1 microliter of Syber Green

After running agarose gel electrophoresis I used the help of the UV light to see if the DNA extraction was done correctly for all except the S. Aureus, and neither of the bacterias travelled within the agarose. When I tried it a second time the results were almost the same, (a bit clearer) but still not one travelled throughout the agarose, only the ruler.

Fresh Start

After a long break, I can honestly say I am glad to be back. I am ready for what is my last semester at Phoenix College, and excited to start my internship with Matt and Josh.

Today was my first day back and because I applied for the MGE Conference for ASU my advisers and I thought it would be a good idea to redo all of my experiments that were done last semester. For those of you who have no idea what my project consist of it has to do with finding the most efficient method for running a PCR. Efficiency is determined by several variables, including cost, quantity and quality of DNA, time required for extraction, and which method produced clear PCR product. In order to be able to run a PCR I first have to successfully extract DNA from bacteria. With that my job today was to make cultures for the several different bacteria that I will be using in both the Quick Extract Bacterial DNA Extraction Kit Protocol and the Isolation and Purification of Total Genomic DNA Protocol.

Rack where Bacteria is stored 

Rack with TSB tubes

Procedures

My four cultures

E.Coli

  Bacillus Agrobacterium S. Aureus

 

Before anything was done, I made sure to label each TSB tube to its corresponding bacteria. The 4 TSB tubes are needed because I have to add a few drops of bacteria in each, helping the bacteria grow. However before adding these drops into the TSB tubes each bacteria was gently pipetted allowing the bacteria to mix, we do this to get rid of the bacteria clumps that are located at the bottom of the tubes. After mixing it well two drops of each bacteria was added to its corresponding TSB tube. These TSB tubes were all covered and incubated. They have to be left in the incubator for a whole night to allow the bacteria to grow in order to use them for the DNA extraction experiments.

Materials                               

E. Coli                                  

S. Aureus

Bacillus

Agrobacterium

4 TSB Tubes

Plan B

In the previous blog I talked about the main goal of my project; to find the most beneficial way to extract DNA from bacteria that will run in two different techniques; gel extraction and in the  PCR (polymerase chain reaction). In the last experiment I was successful in running the gel extraction with three bacterias; Salmonella, E. Coli, and S. Aurues, but not successful in the PCR technique.
The next day after finding out that the PCR did not work, it was time to test a different protocol. This protocol is called "Isolation and Purification of Total Genomic DNA from E Coli", an experiment that is longer compared to my first experiment.
The procedures for this experiment began just like the first experiment. I had to grow bacteria over night for 24 hours in a TSB tube, but since I had my bacterias already growing from the first experiment I went ahead and used the same ones (E. Coli, S. Aureus, S. Epidermidis, and Salmonella). My S. Epidermidis tube was spilled when transferring so I used a different TSB tube that was grown a week earlier. I had to do some centrifuging at high speeds for intervals of 2-3 minutes. As well as some incubating at 37-80 degrees Celcius for 30 minutes and 5 minutes. Lysis solution was added to disrupt membranes and denature proteins. RNase solution was added to degrade RNA into small fragments or ribonucleotides. Protein Precipitation Solution to add insoluble material for the centrifuging step. Isopropanol was added to the solution, also 70% ethanol, and lastly the DNA Rehydration Solution (TRIS EDTA Buffer). In the end of all these procedures the extractions made were ready to run in either  techniques. The first one I did was the gel extraction, the gel was made out of 100 milliliters of TAE and 1 gram of molecular Biology Agarose. When analyzing the gel under uv light, I came to the conclusion that this extraction only worked for the E. Coli and not for the rest. Once again a PCR was not done because I had no DNA from the extraction to run. Here are some pictures enjoy !


Closest to 80 degree Celsius
the incubator reached.  

Gel Extraction Apparatus

TAE Solution

 

Lets do this !

After a week of deciding what my project would consist of, DNA Extraction came to the rescue. I was introduced to the Extraction of Bacterial DNA from Gram-Positive and Gram-Negative species experiment. The goal of this project is to find the best method to extract DNA from various bacteria that will efficiently and successfully run in the PCR (polymerase chain reaction). The PCR is a machine that is used to amplify either a single or a few copies of DNA resulting in the duplication of  several, to millions of copies of a specific DNA section. However to actually run a PCR we have to first extract the DNA and prove that there is DNA present to run it, most of my time was dedicated to this first step of the project. In order to find out if DNA is really gained from the bacteria extraction a gel extraction technique was used to prove it. The gel extraction is a different type of technique that isolates the DNA from an agarose gel, and with the help of uv light we know if DNA is present from the extraction done.

The first protocol I tested was short and simple and glad to say successful. Just a few steps were required as opposed to the second protocol which was more complex. For both, I made a culture for four different bacterias: E. Coli, S. Aureus, S. Epidermidis, and Salmonella. I placed a drop of each into their own TSB (tryptic soy broth) tubes and allowed the bacteria to grow for 24 hours. The next day I noticed that the S. Epidermidis did not have any bacteria growth, compared to the other three. I had to centrifuge each of them for 3 minutes under 5000 rmp, then extracted the TBS. I washed the bacterial cell pellet with.5ml sterile water following up with 3 minutes in the centrifuge. After the pellet was washed I extracted the supernatant and  added 100microliters of QuickExtract Bacterial DNA Extraction Solution. With that I added 1 microliters of Ready-Lyse Lysozyme Solution to each of the tubes. After a few minutes of incubation at room temperature the tubes were ready for the gel extraction and the PCR.


        

Test tubes
Top Row Ready for Gel Extraction
Bottom Row Samples from TSB culture

 

 

Left = QuickExtract Bacterial DNA Extraction Solution
Right = Ready-Lyse Lysozyme Solution


 

My results for the gel extraction were great ! Except for the S. Epidermidis, because just like I suspected the DNA extraction was not successful. You can see this by the really dark square next to the E.Coli. That same day left the PCR running with the salmonella but unfortunately the next day I found out that the PCR test did not work. This conclusion has left me with the second protocol to test. I will soon have results of which protocol gave the best results, and which one is able to continue with the PCR.                                                                  



Gel Extraction under UV Light

First Row                   Ruler

Second Bright Row    E.Coli

Third Dark Row         S. Epidermidis

Fourth Medium Row  S. Aureus

Last Bright Row         Salmonella

Strokes: Therapy, Drugs, & more

 
Restorative Neurology “Drugs and Recovery Following Stroke”

This past week I have dedicated most of my time to an article I found that's main focus was the recovery process of a stroke. The main topic for this journal dealt with four main points, the biological and environmental factors of a stroke, the responses to the injury, neuronal rearrangements, and the adaptive responses.

Thanks to laboratory work, these four factors can now be understood and also provide information on the recovery process for a brain injury such as the stroke.  The way these researches went about their experiment involved four different procedures. Investigations were done for the biological and environmental factors, this was more of an observational study which involves a simple task such as book keeping, and keeping an open eye for changes that stood out. Some of these details that  were compared to each other were for example the size of the specific injury, the location, the rate this injury occurred at and how fast these injuries took to recover. For the responses to injury section and the adaptive responses experiments were done on animals to compare the variety of responses the brain had to different scenarios. Cerebral edema is an example of the most common response, which is the excess accumulation of water in the intra/extra spaces in the brain. These responses were also compared in different areas, how fast they took to respond, how beneficial these responses were, and how they react to a variety of drugs. Taking to note that out of all the drugs that were used the two that are normally prescribed now on humans, actually were the ones that slowed the recovery process even more, these two were phenytoin and benzodiazepines. The third section also involved rats mostly adult rats. The neuronal rearrangements dealt with the new connections our neurons and axons make after an injury occurs and the connections that are lost. Some of these connections appear to happen 8 hours after an injury which would be beneficial if these connections would help, but they seem to impede the recovery process. In the other hand the connections that would be beneficial are of course the ones that take months to happen.

There are many drugs out there at this very moment that seem to aid the injury, however none stand out from the rest. As a person interest in neurology and someone who had her grandpa suffer such an injury I actually found this article very interesting, and even if you don't think this would interest you try and learn a little about it.  I believe we all can agree the brain is the reason we function making it very important, but yet we still do not understand how it truly works.

Here is the link if you are interested in reading more.
http://stroke.ahajournals.org/content/21/11/1636.short

Citation
Goldstein, L. B., & Davis, J. N. (1990). Current Concepts of Cerebrovascular
     Disease and Stroke. Restoratiave Neurology: Drugs and Recovery Following
     Stroke, 21, 1636-1640. doi:10.1161/01.STR.21.11.1636