March+2012

March 2012

Sectioning
So far this month, Francis taught me how to do sectioning of zebra fish eyes. When sectioning, we are looking for eyes that look like this: The dorsal is the top side of the eye and the ventral is the bottom. We are mainly looking at the optic nerve and the retina which is right behind the lens. Though the lens is nice to have to see how close or far you are from the optic nerve, it is not necessary. The lens is the largest when you are at the center of the eye and closest to the optic nerve. Under the microscope the optic nerve appears white and like tangled lines and can be distinctly seen behind the retina.

The machine I use for sectioning uses a knife to slowly cut away at the eye and make thin sliced sections. The sections are called ribbons and I try to fit about 4 or 5 on a slide. The positive side of the slide faces down to attract the ribbon gently to it. Then once it is attached, I can observe it under the microscope to see if it is worth keeping. The slides we want to save are placed on the slide warmer to be used for later. The slide warmer makes the eye adhere more to the slide so that they are safer. Below is a [|slide warmer]:



The eye themselves have to be kept at -20 degrees along with the machine. And they are then frozen onto a chuck which will be inserted into the machine to be sectioned. I have to adjust the chuck so that the fish block is barely being sliced on the blade so that it creates nice even ribbons so that one eye is not being cut at a different rate than the other. This is a picture of what the chucks look like along with what they look like with frozen material on them:

[|Where chuck picture is from]

For my first sectioning, I had two cups of fish eyes that were two different types with two eyes in each. One was labeled Cup 1 and the other Cup 2 ( I have their actual names but they are being withheld) and i set the chuck with them on it so that the optic nerve was facing up and the lens was toward the blade. After getting about 8 good slides of both cups containing the optic nerve on each slide, I placed them on the slider warmer so they could adhere. In both cups, barely any lens was present but there was plenty of optic nerve which is what er are looking for.

Touch Down PCR
We began by first completing a regular PCR reaction for two different combination of primers. Before we ran the PCR, I tested the cDNA to make sure it was good for the PCR by nano-dropping it (see picture below for results). After not too much luck, we decided to complete a Touch Down PCR. A TD PCR annels the PCR reaction as different temperatures so that we can find the most effective one for the primer we are using. For our reaction, we anneled at only 3 different temperatures ranging from low to high. We used the Super Taq PCR protocol: And when I finished adding all the reagents into a PCR tube, Francis told me to put some oil on top to keep everything in the bottom of the tube. It helps when handling the products after the PCR has taken place. By looking at the results above from my nano-drop, we see a peak at around 260nm which is what we are looking for in the cDNA and there is also no other peaks which means there is no contamination in our DNA.
 * ~ Reagent ||~ Amount (microliters) ||
 * < DEPC H2O ||< 13 ||
 * < 10x PCR Buffer (w/ MgCl2) ||< 2.5 ||
 * < dNTP mix ||< 1 ||
 * < F. Primer ||< 2 ||
 * < R. Primer ||< 2 ||
 * < DNA Template ||< 4 ||
 * < Super Taq ||< 0.2 ||
 * ~ Total ||~ 25 ||

Gel
We ran a gel on our TD PCR reaction. In running the gel, I had to remember that DNA is negative so I have to run the gel negative to positive so that the gel will turn out correct. Below was the protocol I used for the PCR: 1. Add Tae Buffer 2. Then add Ethidium Bromide 3. Measure and add 1 gram of Agarose 4. Microwave solution for about 40 seconds, but not so much that it begins to boil over 5. Take 25 uL of PCR product and add 4uL of the dye for the gel. Do for all 3 PCR products 6. Insert about 18 uL of product into each of the wells, skipping a well inbetween each product. 7. Insert the ladder into lane 1 8. Run gel from negative to positive end.
 * ~ Reagent ||~ Amount ||
 * tae Buffer || 100mL ||
 * Ethidium Bromide || 8 microliters ||
 * Agarose || 1g ||
 * Dye for gel || 4 uL ||
 * PCR Product (3x) || 25 uL ||
 * this is the main solution for the gel, once it begins to cool, I then add it into the gel container to harden and I insert a comb to make wells for the PCR product. To quicken the process, I ran the flask under cool water. And then once it has solidified in the container, I remove the comb and pour more buffer over it so the electricity can flow through.
 * it was important that I remembered to break through the oil that I used in the TD PCR reaction so that none of that entered into a well in the gel.
 * When the gel is running, bubbles can be seen to let one know if it is working correctly.

Restriction Digest
We also did a restriction digest of the vector DNA to run on a gel after the digest. I followed the protocol below for the restriction digest we did: 1. Add the above reagents into a microcentrifuge tube in the order they appear. 2. centrifuge the product so that it all sits at the bottom of the tube 3. Incubate in a water bath at 37 degrees Celsius for 2 hours
 * ~ Reagent ||~ Amount(uL) ||
 * H2O || 12.5 ||
 * 10x Buffer (buffer 4) || 2 ||
 * Vector DNA || 5 ||
 * Nde 1 enzyme || 1 ||
 * ~ Total ||~ 20 ||
 * the enzyme has to be kept freezing at all times, that is why it is added last