Post 3

Thanks for following our journey. Since our last post A LOT has happened, so get ready to learn about the good, the bad, and the fun from the past couple of weeks.

The Catastrophe

Don't worry, we figured it out... eventually

After running our first DNA extractions on our own we prepared to run PCR for the first time. This basically means we were going to make a bunch of copies of the DNA to make it easier for it to be "read" when it is sequenced. In order to do this we needed several parts to make the reactions work; a small sample of our extracted DNA, polymerase to build the new strands of DNA, dNTP's which become the new parts of the new strands of DNA, and finally, primers. We made a master mix that included all of these parts and then added our DNA to the individual tubes.

Little did we know, one of these parts would betray our trust...

Above is a picture of our gel which to our surprise showed no DNA in any of our samples.

After we ran our PCR procedure we ran all of our samples in a gel which is used to show if the DNA has actually been copied. Unfortunately for us, nothing showed up in our gel meaning that something hadn't worked.

But what had we done wrong?

At first we asked ourselves if we could have messed up during extracted? we thought that this probably wasnt the case because we extracted half of our samples with our mentor, Dr. Wilson and he definitely knows what he's doing. It had to be something wrong with making copies of the DNA then, during a step called PCR. But what part of the PCR procedure went wrong then? We had no idea, so we had to test every idea and guess if it was the problem.

Could it have been the primers (molecules that tell where to start making copies of the DNA so only the part we want to see is copied)? Maybe, but probably not. The other groups of students in the year one biotech class were very successful in getting results when they extracted the DNA from fungi. That means they were successful using the same primers we were using too, so the chance that our primers were bad seemed pretty slim.

Was it the program for our thermocycler(The machine that is used to make the temperatures of the samples perfect for the DNA to be copied)? Probably not. We had found the program on the website for one of the solutions added to the samples known as polymerase which builds the copies of the DNA, so the program was specifically designed for what we were doing. Below is a picture of our thermocycler program which rapidly cycles from high to low temperatures.

Could it have been the gels we used to see if we had a DNA sample? We used some really fancy gels with a dye that would allow the DNA to be seen already built into them. What if there was a manufacturing error and no dye was added? Then we wouldn't be able to see our samples even if there was DNA present. Below is a picture of how the gels are supposed to look if they work. Unfortunately for us, the gel we used looked nothing like this.

But the polymerase, could that be the problem? Even though it was stored in a freezer kept at a blistering cold temperature of negative 80 degrees Celsius, it was in fact expired. That should be the problem then, or so we all thought. However, it turns out that wasn't it though.

After a week of testing a few of these theories and talking with the teachers we finally found our problem. We made a rookie mistake... The primers we used during PCR were already diluted for another PCR protocol. When we made out "mastermix" we diluted the primers even more, so that the DNA wouldn't replicate. It's pretty safe to say that we will not do that again.

Using the wrong primers for our procedure. Now that, was embarrassing.

Redemption & Bluer Skies

Finally, after many days worrying about a multitude of potential issues with PCR, we gained the correct primers, and began a new methodology. Instead of using a complex series of dilutions and the creation of a master-mix, we gained access to "PCR Beads", a sort of all in one reaction kit, thanks to Rock Canyon. For this, we simply added DNA and our new correct primers, to a small tube containing a bead of all the necessary components for PCR.

Following our successful PCR, we were good to move ahead and finish our extractions. With our methods solidified, there was nothing to stand in our way. And with that, we were back on schedule, and able to complete our research by the end of next week. From this point, we only need to run electrophoresis on the rest of our samples, clean them, and then run a nanodrop to confirm the concentration of DNA. It should be blue skies from here on out.

Above, our self made gel setup with a UV dye in order to visualize the DNA. We called this "Trash Can Electrophoresis," because we needed to cover the gel to keep the dye from activating. Luckily, this method allowed us to confirm that we in fact successfully extracted DNA and completed PCR.

I Walk These Lonely Rows

Camden's Side Quest At The Herbarium

While many people chose to take the week of our Thanksgiving break to rest and recharge (Andrew and Jason left me, Camden, in the snow to go on tropical vacations) I stayed here in Colorado to do some more lab work, as a sort of side quest. We were running a bit behind on our sample collections, so some extra work would definitely help us to get caught up.

I went down to Dr. Wilson's lab on Black Friday, no special shopping deals for me, to collect the rest of our samples and to work on extracting the DNA from some of our samples. That way we would be all caught up on our lab work after "The Catastrophe".

The lab setup at Dr. Wilson's lab.

Once all of the tip cases were loaded I could finally begin the DNA extractions. By now I had extracted a total of 12 samples, so I was starting to get good at this. Sitting alone behind the desk for extraction I got into my zone and got to work. By now I knew exactly how to run this procedure and I was beginning to feel like an expert on it. I barely had to think about the steps to the procedure at this point and just kept moving through the procedure. Of course I still checked my notebook for the procedure just to be 100% sure that what I remembered doing was what I was supposed to be doing.

In no time at all I was finishing up the extraction and collecting the DNA so that I could make copies of it later. In the end it only took me only three and a half hours to run the procedure this time, and I did it completely alone. That was down an hour and a half from the first time we ever used the procedure.

Finally, once I was done collecting the centrifuge tubes with the newly extracted DNA, it was time to head back home and spend the rest of my break relaxing.

Thank you so much Dr. Wilson for letting me use your lab to do our research!

The lonely rows stocked full with mushroom samples at the Sam Mitchel Herbarium

After arriving to the lab I learned that I would now have to pay a tax to use Dr. Wilson's lab. It was time to refill the micropipette tip cases (used to keep pipette tips sterile and easily accessible). Unfortunately, unlike the school's lab with quick loading tips, I had to load each tip individually. Oh well, once it was done I would have access to all of the tools I could possibly need to extract the DNA. Fortunately, I remembered to bring my earbuds, so I turned on some music and loaded the boxes to the beat to make it more fun.

A spin column used to separate the DNA from the other cell parts. The bottom contains some of the cell parts while the DNA is trapped in the white pellet in the middle of the tube.

Avengers Assemble!

Above is a picture of our basic tube rack setup. It includes every micro centrifuge tube and mini column needed to extract the DNA from 8 samples. We found setting up our tube racks like this would allow us to do our work as efficiently as possible.

By this point we were starting to get pretty fast at our DNA extractions and did 24 extractions in about three and a half hours.

After blistering snow storms for Camden, warm Hawaiian sands for Jason, and the charm of Southern Georgia for Andrew, our team reunited after what felt like a millennium away from the lab. The Monday we all met in the lab, we felt stronger than ever. Refreshed, and back on track after Camden's Side Quest and fixed methods, our team embarked upon a daunting task. We sought to extract another 24 samples in one day. We had done it before, but did we remember the methods? Would we be able to finish in time? These questions lingered in the back of our heads, but we knew that the Funguys would prevail, just like we always do. We worked all through lunch, class, and after class, until we emerged victorious, with 24 fully extracted samples. With these samples completed, all that was left of this phase was the last 15 samples. The horizon had never appeared clearer to us. With the primer fiasco in our rear view, we were set up for success.

87 Samples, And an Eternity, Later...

On December 4th we were finally ready to extract the DNA from our last 15 samples. We had spent the previous day getting everything ready so that we could work as efficiently as possible, and all of our work the day before paid off. We started off our DNA extractions as relaxed as we had ever been in the lab. We practically had the entire procedure memorized at this point and were just going through the motions. It seemed that this time we were almost moving too fast, like we were skipping steps, but not a single step was skipped. We were pros at the procedure by now after already extraction a total of 72 samples by this point.

It seemed like just after we started we were already done with our DNA extractions. That was it, the last of our samples. We had extracted a total of 87 samples now and we were so excited to finally be done with this step after working on it for close to 2 months.

Now that we have finished our extractions we can finally move on to the next step... PCR.

"Property of The Funguys!", every single one of our extracted DNA samples from the past 2 months. 87 samples in total. Now that's a lot.

PCR, Our Old Nemesis.

So We Meet Again...

We finished off the week by running PCR on all of our 87 samples. This was in order to make a bunch of copies of the DNA so it can be sent off for sequencing. This time though, we used the right primers for our PCR method. In this case, we used what is called PCR beads which include all of the things that are needed to perform PCR all wrapped up in a little dehydrated bead that just makes life easier. All we have to add a little bit of our samples and the primers (the right ones this time) to tell what part of the DNA we want copies of.

In a way, this is much more efficient than what we were doing before.

The Office.mp4

Will PCR work? Did we actually extract the DNA to begin with? What species will we identify?

Join the Funguys for our next post on January 24th for the Grand Finale of Phase One of our Research!

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