EUREKA!!! (almost)

The PCR process was successful! Well, sort of… As is seemingly the norm for me, my results introduced some new questions. I’ll get to that in a moment. First and foremost, when electrophoresis was conducted using my PCR results, there were evident bands of DNA that traveled in the gel. This means that even though there was sheering evident in my extraction method, there was a viable product that yielded suitable fragments of DNA that could be amplified through PCR.

The chloroplast primers I used were not suitable for purposes of differentiating between the individual test subjects of my research. These primers are very general in nature and were just used to see if the extraction results contained amplifiable DNA. This is the Eureka part. The new questions arise from where the bands occurred on the gels.

PCR was conducted with the results from three different extractions: the 1^st was the extraction that appeared to have a massive amount of DNA with heavy shearing, the 2^nd was the same protocol but done in a linear manner (in the 1^st the protocol the run was broken up due to time constraints, and this may have caused shearing), and the 3^rd was the same as 2^nd run, with the addition of using improvised wide bore pipette tips (again to try to reduce shearing). Each sample went through PCR using 3 different concentrations of each extraction for a total of 9 PCR reactions. The banding appeared in the wells containing the 1μL and 2μL extraction concentrations. There was no banding/travel exhibited in the 3μL concentrations. To relay a clearer version of what I observed in the 3 gels, I reran in a new gel omitting the 3μL concentrations. Below is the image of that gel.

As wonderful as it was to observe a discernable band of PCR results travel in a gel, my elation was cut when I noticed where the travel occurred. Evident bands appeared in two different extractions at two different concentrations! Even though I was extremely meticulous in labeling, handling and loading the samples, it is my hope that I mislabeled or misloaded one of the samples. I cannot come up with a viable explanation for these results – it just does not make any sense.

It appears as though next week I will give it another run…

PCR ASAP

Well, as I feared, the "no grind" extraction was fruitless. The "wide bore" extraction was unfortunately not a resounding success either. There was DNA, but it appeared to be sheared (again). Another interesting point to note, is the distance the samples traveled through the gel. As far as my understanding goes (and from many, many conversations with people more in the know than I), genomic DNA typically does not travel as far as it did in my gels. Genomic DNA is relatively large and therefore should not migrate very far in the gel at all. Could the additional observed travel be a result of shearing making the molecules smaller and therefore easier for them to travel in the gel?

As the time crunch is officially upon us, I have decided to see if, within the potentially sheared DNA samples, there is something the chloroplast primers can identify and reproduce. It is sort of a Hail Mary at the end of the semester in order to see if I can get some form of PCR results to use in comparison of the individual subjects.

For PCR, I mixed 550µL of master mix with 11µL of chloroplast primer to create a stock solution. 20µL of this stock solution was added to each PCR tube. Then, I added a combination of DNA extraction sample and Ultra Pure H₂O totaling 20µL. For each of the three DNA extractions I conducted 3 PCR reactions: tube one held 1µL of sample, and 19µL of Ultra Pure H₂O; tube 2, 2µL of sample, and 19µL of Ultra Pure H₂O; tube 3, 3µL sample, and 17µL of Ultra Pure H₂O. The protocol or pattern for the PCR cycle is as follows:

1. Initial Denaturation: 94°C for 2 minutes
2. Denaturation: 94°C for 1 minute
3. Annealing: 59°C for 1 minute
4. Extension: 72°C for 2 minute
5. Steps 2-4 repeated 40 times
6. Final Extension: 72°C for 10 minutes
7. Hold: 4°C indefinitely

Tomorrow I will cross my fingers and run another gel with the PCR results. Here's for hoping!

Preparing the samples for PCR.

FIELD TRIP!!!!

Last Friday was the S-STEM field trip. We journeyed out to Dreamy Draw to take look into the ecology of ephemeral washes. Very familiar territory for me both literally and figuratively. The location was almost on the exact opposite side of Squaw Peak from my sample collection site, and my collection site also happens to be an ephemeral wash! The dry washes are a very interesting phenomenon ecologically speaking. As I am currently conducting research in a one of these washes, I have previously discussed much of the information that Matt relayed to us concerning them.

We also did a fair amount of geocache hunting. I really enjoyed this it had been at least 5 years since I have done any geocaching, and I am wondering why I ever stopped. In addition to this, Josh and Jenni both planted a couple of geocaches. I had never done this previously. Good times! Josh placed his at a fork in the path and had a clever clue to it's location based, of course, in Star Wars. Jenni found a large hole that turned out to be what we think was an exploratory mine. This area was used in mining cinnabar (mercury).

Matt, Josh, Andrew, and Jenni in Jenni's hole

As far as my lab work goes, This being a holiday, I don't have a lot to report. Monday's lab time was spent examining protocol for the sucrose based extraction. Today I conducted the PCI extraction on a fresh sample as well as the sample I soaked in the lysis buffer without grinding. Both of these instructions were completed using my makeshift wide bore pipette tips in an effort to reduce shearing. At the moment both sa,mples are held in a proscribed overnight freeze. Tomorrow, the extractions will be finished. As of now, the fresh extraction appears to be promising - I am seeing a precipitate (DNA) come out of solution. The other extraction, not so much. The solution is VERY clear - no signs of precipitate at all. Negative results are still results...

Still plugging along

This week I conducted another run with the phenol chloroform extraction. In this attempt, I halved the amount of initial plant tissue, and worked to reduce the break points in the protocol. This being my second usage of the protocol coupled with my conducting it on a day in which I had more hours to devote to it, allowed me to conduct the protocol seamlessly with no breaks other than incubation time. Unfortunately the results still exhibited signs of shearing.

This is a picture of the gel from the camera mounted on the UV light box. The beginings of banding can be seen, but the DNA sample is sheared (the lanes are just streaks)

I am currently working on two other ideas that will hopefully reduce the shearing. The first step of the protocol is to grind the leaf tissue samples (-80° C) in the lysis buffer containing the 5% sarkosyl solution and the 4% SDS solution, and then incubate it at 50° C for two hours. It is my suspicion that shearing may be occurring in the grinding. I have taken tissue samples and incubated them in the buffer for an extended period of time without grinding. I am not sure if this will yield anything, but it does merit testing. The other potential shearing point that I see is the pipetting of the samples. I suspect that drawing the samples through the narrow tip of a standard pipette could cause shearing. Since there are not any wide bore pipettes available for use, I will snip the pipette tips back to a wider opening for each pipetting. I had intended on this today, but unfortunately the BIO 181 classes are using the fume hood today. Normally Tuesday and Thursday are the days I have enough time in the lab to carry out the full extraction. Next Tuesday is a holiday, so I will not be able to revisit this until Thursday. Hopefully I will see a clean nicely banded lane in the well

As to the Sucrose extraction that Matt clued me in on –

I have read that this is to induce an osmotic shock within the cells as a means of cell lysis. The addition of the sucrose in the solution increases the solute concentration outside of the cell. This rapid change of concentration results in an equally rapid change in the movement of water across the membranes (osmosis). The drastic change in pressures should lyse the cell. If this is actually what happens, I should be able to use a sucrose lysis buffer and not have to manually grind the tissues. This is in the same vein of what I am doing with the current extraction – attempting to lyse cells with out physical tissue homogenization. There was one protocol I read that specifically stated that the sucrose buffer was very effective for plants that had been previously shown to be difficult to extract DNA from. Unfortunately they didn’t say why. I am still digging to see if I can find more information on this.