Week 14-Research On Hold

Today is the final week of activities for this course, so any further research is on hold until the summer session begins. I have plans to repeat my methodology and protocols, double-checking calculations to see if I can determine what errors occurred that resulted in sheared DNA.

For the remainder of this week, I will be busy presenting at the Estrella Mountain conference, completing my research paper, and doing a brief oral presentation on my project for the other S-STEM scholars and Bio-Sciences department staff.

I was briefly disappointed, when I realized that my research to-date had been unsuccessful, but I very quickly got over it. I now view the project as incomplete, instead of failed, and will strive to complete it prior to the beginning of the fall semester. While I wait for a continuation, here is something for you to enjoy- My gels should have been horizontally banded like this image (minus the coloration), but were not:

Credit: www.helys-dna.com

Week 13- Elusive results

Last week, I was able to run another PCR and electrophoresis gel on my E. coli DNA samples. This time, I changed the primers that I paired to two that had melting temperatures with 4°C of each other, due to the prior PCR's annealing temperature being too low to generate results.

These are the primers that I used: 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). They had melting temperatures of 59.4°C and 55.8°C, respectively, so I ran the PCR with an annealing temperature of 54°C.

Unfortunately, while there was DNA present in my gel, it was sheared and did not successfully amplify. Here's a screen capture of the first gel from last week:

And here is the secondary image with the higher annealing temperature of 54°C:

As you can see, both gels are lacking the clearly define horizontal bands that indicate the presence of amplified DNA. So I will have to refine and repeat my methodology as I proceed. There is good news, however- I have been approved to return as an unpaid intern over the summer session. I will be able to continue my research. I am doing this for the sheer joy of the learning involved, and also so that I will hopefully be ahead in my project with the onset of the fall semester.

Week 12-Limited results

Today, I ran an electrophoresis gel on the six PCR samples that I created last week. Once again, I was reminded that a hurried project is a failed project, as I had to load three gels due to a calculation error in my sample sizes. The error occurred as a result of me attempting to rush through the loading process, instead of taking the time to check and then re-check my samples.

While the PCR process worked, it was not at optimal amplification and my gel was muddled. The likely culprit for this was probably the annealing temperature, which was set for all six samples at 43°C. The melting temperatures for my primers ranged from 48°C to 60°C. I had set the temperature to accommodate the lowest melting temperature of my four primers, in order to run all six samples through the thermal cycler during one PCR. Next week, I will repeat the PCR, but at that time I will run separate cycles depending on the primer samples, so that I can more closely align the annealing temperatures with the utilized primers.

Here is an image of the gel results:

Week 11- Turning up the heat

This week I began constructing the rough draft of my research paper. While incomplete, the formatting has been done and the paper is ready for insertion of data as the project progresses. Given the nature and pace of the project thus far, I anticipate final completion of the project to occur in the fall semester. That said, I have finished the initial phase of the project, which dealt with testing various DNA extraction protocols. I tested four protocols that utilized boiling, digestion, and cold ethanol. The simple boiling methods worked best not only at extraction, but were also preferable for ease of use. As one of the goals of my project is to develop a micro lab that will introduce the student learner to elementary DNA identification, it is important that I identify a protocol that is both simplified and time-efficient, so that the method will fit in the typical allotted class time.

This method was both easiest to use and successful at extracting Escherichia coli DNA:

 

Protocol 1: Autoclave 200 mL of de-ionized (DI) water on media cycle @ 121°C/15 PSI. Centrifuge 400µl of E. coli culture @ 10,000x g for 10 minutes to pellet the sample. Re-suspend pellet in 100µl of autoclaved DI water. Incubate for 15 minutes @ 100°C in hot plate bath. Centrifuge sample @ 10,000x g for 10 minutes. Remove supernatant and place into new Eppendorf tube. Store prepared sample @-20°C until ready to use.

This protocol had the added benefit of teaching the student sterile technique, due to the utilization not only of the autoclave, as in P1, but also the use of molecular-biology grade water:

Protocol 4: Autoclave beaker on media cycle @121°C/15PSI to sterilize. Add 1ml of E. coli to 1.5 ml Eppendorf. Centrifuge @13,200x g for 15 minutes. Eliminate supernatant, and re-suspend pellets in m-b grade water. Centrifuge @13,200x g for 10 minutes. Eliminate supernatant, and re-suspend pellets in 40µl of m-b- grade water. Boil @100°C for 10 minutes. Cool on ice. Centrifuge @13,200x g for 10 seconds.

Today, I begin DNA amplification via the PCR process, so I will begin testing the universality of the primers I chose for this project. More on that next week. Cheers!
Here's a PCR machine in the old days, and a link to its origin and some useful information:
http://www.plantcellbiology.com/2012/03/you-dont-know-how-lucky-you-are-pcr-in-the-old-days/

 

 

Week 10

This week brought the news that my research project was accepted for presentation at the Estrella Mountain Student Conference; the S-STEM scholars, including myself, will also be presenting at Metrotech high school on April 25th. I look forward to the opportunity to hone my research for presentation to both groups. Truth be told, I am more interested in presenting to the vocational students than the other undergrads, as I look at it as an opportunity to potentially open a student's mind to the possibility of higher education.

My initial work in the lab this week was focused on completing an extraction protocol on the E. coli DNA that I have been using during this first phase of my project. Then I used a Promega Wizard DNA clean-up kit to remove contaminants from all four samples of DNA that I extracted last week. The cleanup protocol is as follows:

1. Re-suspend clean-up resin by inversion.
2. Add 1 mL of the re-suspended resin to a 1.5 mL micro centrifuge tube.
3. Add the sample to the clean-up resin and mix by inverting several times.
4. Attach a micro column to the barrel of a sterile 5 mL syringe.
5. Pipette the clean-up resin containing the bound DNA into the syringe barrel.
6. Slowly and gently push the slurry into the mini column with the syringe plunger.
7. Add 2 mL of 80% isopropanol to the syringe and gently push the solution through the mini column.
8. Transfer the mini column to an eppendorf tube and centrifuge for 2 minutes at 13200X g.
9. Transfer the mini column to a new eppendorf tube and apply 50 µl of 1x TE buffer and wait one minute.
10. Centrifuge the mini-column for thirty seconds at 13200x g.
11. Discard the mini column and keep the eppendorf tube containing the supernatant. The prepared sample can be stored at -20°.

I then prepared all four samples for an electrophoresis to identify if DNA was extracted. I used a 1x TAE solution to prepare both the buffer and the gel, then added 2 mL of each sample separate wells, followed by 1 mL each of loading dye and SYBR green. After adding the buffer, I initiated the ep process and covered it with a lab coat to reduce light interference. The gel ran from 24 minutes. The following is an image of the completed gel, with the two wells to the left consisting of control DNA provided by the lab coordinator, Josh.