Busy Week

During last week while almost all of the other inters were working hard on their posters and getting prepare for their presentation for ASU conference, I had a busy week. I did lots of different things for my project, but now I feel that I didn’t do any useful thing in the last week.  On Monday, I decided to set up another PCR from my purified Agrobacteria’s DNA to extract it with Agarose Gel Electrophoresis technique because I was not able to get a good result. I used three different primers to prepare my PCR (Polymerase Chain Reaction). When I finished it, I found out that  PCR machine would be used by one of the Biology classes for their lab for a whole week and I would not able to use it. Then I incubated my PCR tubes in the refrigerator at temperature -4 degree C to do PCR in the next week. On Tuesday, I cultured my Agrobacteria in the three different kinds of bacterial growth mediums. I used TSA (Trypticase soy agar), YEB agrobacterium, and MacConkey’s (MAC).  Then, I place them in the incubator at temperature 37 degree C.Another interesting thing that I did on Tuesday was planting 10 soy beans for my experiment. Now, I feel that my experiment is getting more serious.

 On Wednesday, I checked my bacterial growth mediums and Agrobacteria grown in the TSA and YEB medium but it did not grow in the MAC. I placed back MAC in the incubator for 24 more hours. Then, I decided to do gram staining my Agrobacteria which grown in the TSA and YEB mediums.  The Gram stain is one of the common methods for staining procedure in microbiology. This method is very useful in identifying bacteria.

 

 At first, I made a smear of Agrobacteria then I started gram staining. first add the crystal violet to the sample on the slide and leave it for 30 seconds, and then rinse it with distilled water. Then add the Gram iodine for 1 minute, and rinse the slide with water again. Next we need to use a Gram decolorize for almost 5 seconds and rinse it with water after 5 seconds. It decolorizes the sample if it’s a Gram negative and it will remove the crystal violet. For the last part, add the Safranin to the slide and after 30 seconds rinse the slide with distilled water for almost 5 seconds. If it’s a Gram positive bacteria, it keeps the crystal violet and it looks purple under microscope.At the end, i viewed my slide under a microscopic and took a picture of it. Pictures of Agrobacteria in both mediums show that Agrobacteria is gram negative.

New Protocol for DNA Isolation from Bacteria

I used new kit to isolate my Agrobacteria's DNA during last week. New Kit has a simple and easy way to isolate pure DNA cultured bacteria. The kit contains all of the reagents needed to isolate and purify genomic DNA from gram-negative bacteria.

At first, I started to dilute Wash Solution Concentrate. I added 80 mL of ethanol 90%  to the Wash solution bottle which contained 10 mL of solution. The next step was preparing Proteinase K for the experiment. I dissolved Proteinase K powder in a bottle in 0.5 mL of Water RNA/DNA.

For the first step, I poured 1.5 ml of 18 hours old Agrobacteria into a centrifuge tube and centrifuged it at 12,000–16,000 × g for 2minuts. After centrifuging, I pipette out the culture medium completely. Then, I added 180 μL of Lysis Solution T and 20 μL of RNase A Solution in the tube. After 2 minutes incubating at room temperature, I Add 20 μL of the Proteinase K solution to the solution. Then, I placed tube into a 55 °C water bath for 30 minutes. After that, I added Add 200 μL of Lysis Solution C into the centrifuge tube and vortex it for 20 seconds to have a homogeneous mixture and  I put it back to the 55 °C water bath for 10 minutes. Then, I Add 500 μL of the Column Preparation Solution to each pre-assembled GenElute Miniprep Binding Column which is seated in a 2 mL collection tube and I Centrifuged it at 12,000 × g for 1 minute. The eluate was discarded. For the next step, I Added 200 μL of ethanol 90% to the solution and vortex it for 5–10 seconds to have a homogeneous mixture. I transferred my solution in the centrifuge tube into the binding column and centrifuged it for 1 minute at ≥ 6500 × g. Then, I placed column in a new collection tube. Then, I added 500 μL of Wash Solution 1 to the column and centrifuged tube at ≥ 6500 × g for 1 minute and I placed the column in a new 2 mL collection tube. After that, I added Add 500 μL of Wash Solution to the column and centrifuge for 3 minutes at 13000 × g. After centrifuging, I emptied the collection tube and put a column back on it. Then, I Centrifuged the column again for 1 minute at the same speed. The collection tube was discarded and column was placed in a new collection tube. At the end, I added 200 μL of the Elution Solution to the column and centrifuged for 1 Minute at 6600 × g. for getting better result of elution I repeated this step one more time.

I used this purified DNA to run a PCR and an agarose Gel.

First DNA extraction

  Second DNA extraction

In my first DNA extraction, I used isolated DNA to run an agarose gel and for the second DNA extraction, I used PCR of isolated DNA.

Gel Electrophoresis of DNA

I learned how to do DNA Extraction during the last week. I used Agarose Gel Electrophoresis technique to for the separation of Agrobacteria’s DNA fragments. The result of Gel Electrophoresis is the separation of charged molecules. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose.

Electrophoresis is a method used in the laboratory that results in the separation of charged molecules. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose.

 At first, I made 1% Agarose which was contain 1 gram molecular Biology Agarose and100 ml 1x Tris-acetate-EDTA (TAE) buffer. After mixing Agarose powder with TAE buffer, I heated my solution in a microwave oven until completely melted.

Then, I poured hot Agarose solution into a casting tray and placed a sample comb into the top part of tray and allowed Agarose to cool down and solidify at room temperature. Meanwhile, I tried to make my DNA samples. I set up 1 Ruler and 4 samples for Agrobacteria’s DNA.

DNA Samples
Agrobacteria's DNA	7 µL
5x Orange G Loading Dye	2 µL
Sybergreen	1 µL

Ruler
PCR MW Ruler	9 µL
Sybergreen	1 µL

 When the Agarose was solidified, I lifted up the comb gently. The comb was created several places on the gel to load different samples.

I poured TAE buffer into the gel running tank and it should cover gel surface. Then, I tried to load my samples. I loaded 10 µL of the Ruler with a Micropipettor into first well from left side. Then, I started to load 10 µL of four Agrobacteria’s DNA samples with a Micropipettor into each well of my gel.

When my gel was prepared and loaded, the electrical leads on the running tank were connected to a power supply. I connected the leads in the way that the red (positive) lead is at the end of the gel to which the DNA will migrate and the black (negative) lead is at the end of the gel containing the wells. Then, I set power supply on 120 V and allowed my gel to run for 60 minutes and I placed a bucked on top of the gel running tank because the gel has to run in the dark.

After 60 minutes, I turned off the power supply and unplugged the gel running tank. Then, I removed our gel from the running tank and carefully carried the gel to the ultraviolet light-box. Because gel holder was not UV transparent so I placed the gel onto the glass surface of the light-box. The DNA bands in the gel light up when exposed to UV light. The box exposes the gel to UV light. I took a picture from my gel.