What I do all day

Today begins my Thesis Project. Basically, if my efforts over the next 10-12 weeks bear fruit, I might get to graduate.

I'm investigating whether the intra-host quasispecies (genetic variants of the same virus) has a different make-up in acute Taura Syndrome Viral infections of shrimp vs chronic infections. Chronic stages are less virulent, they represent an equilibrium of sorts in the animal where they are still infectious, yet they don't die.

To relate this to humans, Hepatitis C is studied in much the same manner, and the quasispecies makeup IS different in acute vs chronic HCV infections. (Reference to be cited latah).

So.. first we injected some shrimp with the disease last Thursday. The buggers began to die yesterday. So, I have samples:



They are samples from 4 shrimp, in duplicate except #4 I grabbed and thawed only 1 so eh thats enough.

I suspect #3 is not infected, because its hemolymph clotted. TSV infected hemolymph usually does not clot.

Step 2 coming soon..

I found a protocol to test for TSV infection

I found a protocol that calls for PCR (Polymerase Chain reaction) of a diagnostic gene sequence for TSV that uses hemolymph (blood) directly, without needing an extraction step.

Because I am leery of New Things.. I decided to both extract the RNA from the blood AND try the protocol with the blood itself.

Molecular Biology is mostly like cooking with Hamburger Helper.. you provide the hamburger (the sample) and the rest is all contained in a handy little kit. Except instead of costing $0.69 at Wal-Mart, this one is $200 from Roche.





It does 50 extractions and all you have to provide is the 100% Ethanol. Partay time.

Well.. the protocol is straightforward enough, except that I screwed up and forgot a step. As a result my yields sucked, as read in the handy dandy spectrophotometer that reads absorbances at 240 nm to tell me RNA concentration in a sample.

I tried the PCR anyway, its currently cooking in the handy dandy Thermal Cycler, which is really a glorified water bath capable of fluctuating temperatures automatically (ie it can cycle 95 degrees for 45 sec then 54 degrees for 30 sec then 72 degrees for 30 sec.. repeat 40 times. ) Doing PCR manually would suck ass, and I am grateful I wasn't in grad school when it had to be done that way.



It has about 10 min left to cycle, and by then my gel should be hard.

Oh yeah, the fun part. Gels.

To visualize the DNA fragment you just amplified (or didn't.. as the case may be if the sample is negative or if your sample sucks or if the Gods are not smiling that day) in end point PCR you make a high tech jello mold, using agarose and buffer instead of jello and water and spiking it with a chemical called ethidium bromide to bind the DNA and make it glow in UV light.




The gel is made just like jello. Mix the powder (agarose) and buffer, heat it in the microwave till its clear, then add the EtBr and pour in the mold and wait to harden. Takes about 30 min.

Once its hardened, you add a loading dye to your samples to allow the liquid to sink into the little wells in the gel. Then you load the gel by squirting about 10 microliters (1000 microliters in 1 mL - so its about the size of a drop of water) into each well.

Ok, my samples are ready for this now so off I go!

Just loaded my 18 samples into the gel. Now it has to run for 30 min or so then I will go read the results. Here is what it looks like when samples are in the gel ready to go.



Then we plug it up to the power supply, crank it to 300V (I love using sodium borate buffer... can crank it to 300V and have clear, readable gels in 30 min vs TE buffer that can only go at 150V or so and takes an hour).



The plastic sheets are to cover the gels while they run.. I found that running them under the flourescent lighting in here bleaches them out and its hard to read them.

So, now I goof off or work on sending out some protocols to a shrimp farmer in Brazil who needs to test his broodstock for my repertoire of diseases. Too bad they can't fly me to Rio to do it myself.

The dye you add in to the samples also serves a function of showing how far your samples have migrated. DNA travels from the - (black) to the + (red) terminals with the current.





The yellow line is about how far its gone. At this rate I'll be home by about 8pm central time. Grr.

Good luck.

Do the "Appease the Molecular Biology Gods dance for me"

I'm already betting it wont work. I guess its worth a peek.

Glowy bands means that there are lots of DNA fragments there.. all match the correct size I'm supposed to have. Except 1 negative but that could be the test gone awry or something. I'm happy with this many +s.

A more formal, scanned image of the gel result. The first and last rows are a molecular weight marker, Rows 2-8 are my samples using whole blood (I didnt think it would work) 9 is a positive control using a known + extract from another experiment, 10 is a No Template negative control, 11-17 are the same samples but using extracted RNA as a template for the rxn, 18 is a positive control, 19 a negative control again and 20 is the molecular weight marker.

Now I can go home

.. and continue this tomorrow or Thurs. Next Step: Amplify my capsid gene now that I know TSV is indeed there. (I don't want to waste the more-expensive reagents till I did this first to be sure its there.)

are you allowed to release this information? .... I don't know, I'm just making sure you're not going to get in trouble for some weird reason.

Since it isnt an official writeup, I don't think it will count as a publication. Some journals won't take data that has been released somewhere else. But since I didnt give any real info, just a very generally vague overview I think its cool.

I can't put the actual paper online though before publishing it or it will count as "previously published data".

Why are those men in black trenchcoats preventing me from leaving the lab. AIEEEEEEE!

*hides*

j/k.