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