Summer Session-New Primers Tested

Hello! Welcome back!

This week, I tested a set of primers which I designed last spring but had not yet used in a PCR. These primers, which I call P-CAT-3f and P-CAT-3r, were created during the same sessions that produced the P-CAT-4 primer set which I have been using to amplify DNA this summer.

As you may recall, the primer set sequences I am using were identified after I analyzed the 16s ribosomal gene maps of three gram negative bacterial strains which we have here in the lab. I then began this phase of my project by testing one primer set against nine gram negative species, including the original three used to design the primers. When several PCRs were run and DNA was amplified, I then tested the primers against six gram positive species, which also resulted in DNA amplification.

For this week's test, I first performed an extraction on the six gram positives I am using, then ran a PCR using the aforementioned P-CAT-4 set. After running a gel and verifying that DNA was present, I then took the original extraction sample and set up a new PCR using the P-CAT-3 primer set.

The results? Five of the six species amplified. The sixth, E. faecalis, did not (see gels below). Base pair sizes were approximated at 350-425 BPs long.

That's it for this week. Until next week, please enjoy this lecture from Dr. Jennifer Doudna, who co-discovered the CRISPR gene editing technique. Cheers!

https://www.youtube.com/watch?v=SuAxDVBt7kQ

Gel results:

 

Summer Session-Material Review

Hello! Welcome back to my blog. This week, I spent most of my time learning more about the technology I am using and reviewing various published papers on the nuances of the 16s gene. However, I did manage to get some time in the lab; I ran an experiment to test the effect of a lower agarose gel concentration on visual review of DNA samples under UV light. Thinner gels can increase the speed of particle travel during an electrophoresis, but what I was particularly looking for was a reduction in the "warbling effect" I have been seeing in the sample banding of my 1% gels. So I changed my concentration to 0.7 % and ran an electrophoresis to measure the results. The gel concentration did not make a difference in the banding or migration rate of the samples I tested.

From there, I moved on to running a PCR on my gram positive bacterial species using primer set 4-CAT-f and 4-CAT-r. I just wanted to repeat some earlier data by replicating previous methodology, so I cultured a new set of bacteria, performed an extraction, ran the PCR and gel-tested the results.

From the pics I took post-PCR, it appears that amplification was again successful. This is a good turn of events, as it allows me to proceed with the next phase of my project. Namely, sending my samples out to be sequenced so that I can determine which region of the gene was targeted. More on that in a later blog.

Until next week, live long, and prosper.

Photo: PCR samples; pic shows ladder in well to the left; remaining six wells correspond to gram positive bacteria used in this study.

Summer Session-PCR Results for All Targeted Species

Hello! Thanks for joining me for another installment of my blog. Your attention is very much appreciated!

This week, I continued collecting results data for my study by running another PCR on my extraction samples. This time, I decided to attempt amplification on all fifteen of the bacterial species I have been working with (a list is included below). This would be the first time I have run a PCR on all of my gram negative and gram positive samples at the same time, although I have done them in separate batches previously; I did a concurrent test this week so that I could compare the results against those obtained earlier in this project.

I also included two separate samples of P. aeruginosa that were obtained using different extraction protocols, as detailed in an earlier blog post. The purpose of this nuance in my test was to ensure that all PCR conditions were identical, so that any variance in results could be tied to the differing extraction protocols.

The results? DNA amplified; all samples appeared to be approximately 150 base pairs long. (Only two gel pictures representing eleven samples are shown due to a camera malfunction in the lab equipment.)

List of bacterial species used: S. enterica, S. sonnei, P vulgaris, P. mirabilis, P. stuartii, S. marcescens, P. aeruginosa, E. coli, K. oxytoca, B. subtilis, S. aureus, B. cereus, S. epidermidis, E. faecalis, and M. luteus. 

Summer Session-A Little Housekeeping

Hello! Welcome back.

This week, I reversed course a little and cultured some Pseudomonas aeruginosa; I then performed an extraction on this little gram negative biofilm-former using my gram positive extraction protocol, just to see if it would succeed at producing DNA. Why? You may recall that this strain was the only gram negative strain I had difficulty extracting DNA from last semester, and you may also recall that my primers were unsuccessful at amplification during last semester's PCRs as well.

After performing the boiling extraction, I ran my standard gel protocol on the sample and was pleased to find that I had visible banding present under UV. Picture below:

(Photo: Four wells, each with P. aeruginosa; wells show different concentrations of sample.)

This was good news, of course, because it allowed me to proceed with a PCR using the primers I have been testing. To date, all of the bacterial species I have PCR-tested using this particular primer set have resulted in amplified DNA post-extraction--- except for P. aeruginosa. This negative result has been gnawing at me for some time, so anything that gets me closer to wrapping up that loose end is a positive, welcome turn.

So I ran a PCR and then checked the sample using my standard gel. The photo below is what resulted.

So it appears that I got DNA. It's not pretty, but given that I blew up the cells using a boiling extraction, that is to be expected.

That's it for this week. See you on my next blog!

Summer Session-Early PCR Success

Hello! Welcome back.

Last week, I got promising results after testing two simple thermal DNA extraction methods on my gram positive species. The gels I ran post-extraction were positive for DNA banding.

Protocol #1 utilized an SDS/TBS mixture and boiling to lyse the cells and took approximately 45 minutes to set up. Protocol #2 used sterile water and boiling and took about 20 minutes from start to finish.

Bolstered by the results from both tests, I took the DNA samples obtained using protocol #2 and decided to run a quick PCR on them using a primer set that I designed last semester. As you may recall, I was able to amplify DNA from 8 out of 9 targeted gram negative species using a primer set of my own design; this experiment capped off my work last semester.

I fully did not expect to get positive results from this PCR, as my primers were designed using three gene sequence maps specific to three of the gram negatives I previously used for this study. However, after repeating the reaction using six gram positive species, DNA was successfully amplified. The gels I ran post-PCR are shown below.

This is great news! I can now move forward with the next phase of this study. Details on that to follow in a later blog.

Until then, live long and prosper.

Top: Gel 1 Bottom: Gel 2. Both gels were run using the same DNA samples. There are 6 wells loaded; from left to right the species are B. subtilis, S. aureus, B. cereus,S. epidermidis, E. faecalis, and M. luteus. Gel 1 used 2 µl of loading dye and 2 µl of sample; gel 2 used 2 µl of loading dye and 4 µl of sample.

Summer Session: First Protocols Tested

Hello! Welcome back to my blog.

This week, I began testing DNA extraction protocols for gram positive bacteria. After reading several research papers that purported in the abstract to contain a quick and simple method for extraction, I found that these postulations were not simple enough for my purposes. Therefore, I decided to create my own protocols and test them.

Protocol #1 involved pelleting down 500µl of culture (after incubating in TSB for 24 hours @ 37°C) and then re-suspending the pellet with a 30 second vortex in a 300µl TBS/15µl 10% SDS solution. I then allowed this to sit at room temperature for 20 minutes, followed by incubation in a water bath for 10 minutes @100°C.

Protocol #2 involved pelleting down 500µl of culture (after incubating in TSB for 48 hours @ 37°C) and then re-suspending the pellet with a 30 second vortex in a 500µl sterile water solution. I then incubated the sample for 10 minutes @ 100°C.

For each protocol, after incubation @ 100°C the sample was centrifuged @12,000x g for 3 minutes. The supernatant was poured off into a 1.5 ml Eppe tube and the pellet was discarded. 4µl of supernatant and 4µl of loading dye were then loaded into 10% agarose gels with 4µl SybrGreen DNA stain added to the agarose solution before setting the gels; gels were run @100 volts for 25 minutes.

Six strains of bacteria were tested for each protocol. They were: S. aureus, B. subtilis, S. epidermidis, B. cereus, Micrococcus luteus, and E. faecalis.

Visual results under UV light indicated that each protocol produced DNA, with a larger amount present in the samples taken using protocol #1. (As I am posting this blog from offsite, however, I am unable to upload the jpegs to this blog. Gel pics to follow on a later edition.)

These results are promising for a first attempt. I will be refining and re-testing these techniques when I return to the lab next week in order to duplicate my results and potentially increase my DNA yield. Until then, have an excellent weekend.

Photo: M. luteus after gram-staining. Source: Microbeworld.org

 

Summer Session-A New Course of Action

Hello! And welcome back to my blog. This post marks the beginning of my enrollment in this summer course. I was unable to blog for the past month, due to late enrollment. To catch you up on where I left off back in May:

I was in dire need of a break after the busy spring path which I had plotted for myself. After months spent tutoring three classes, mentoring the college prep class at Carl Hayden High School, interning in the lab, and working my job at the Arizona Center for Nature Conservation (The Phoenix Zoo), I decided to work full-time and loaf around while I waited for the enrollment email from Phoenix College. I got to spend a lot of time with my crazy dogs, Mr. Bentley (my pit bull) and Samuel Marshall the Third (my labradoodle). I also squeezed in a bunch of relaxation time, as well. But now it is time to get back to work. I am looking forward to it!

I have decided to move my research project into a slightly different direction. To date, I have been working solely with gram-negative bacterial species. Last semester, I tested multiple techniques for extracting DNA from gram negatives and tweaked every aspect of my project so that each procedure would maximize results from that particular type of bacteria. Even the primers which I developed for use during PCR amplification were based solely on the available 16s ribosomal gene maps of gram negative strains.

This initial focus, of course, was due to the fact that gram negative cells are easier to lyse because of their cell wall structure. Gram negatives, despite having a double-membrane, contain only a thin layer of peptidoglycan between the two membranes. This small amount of peptidoglycan can make cell lysis and DNA extraction easier; that ease of cell disruption can also lead to sheared or destroyed DNA when an extraction is conducted using a lysing additive that is too harsh for this type of cell wall. As a result, I had quite a bit of testing to do of various protocols before I settled on the simple thermal protocol using an SDS/TBS mixture which I described in last semester's research paper.

As I have at this point repeatedly extracted and amplified DNA from gram negatives, I am extending the project to include gram positives. Gram positive cells essentially contain a single cell membrane surrounded by a thick layer of peptidoglycan. This thicker layer (than that which is contained in gram negatives) can be difficult to lyse during an extraction. I have tested my gram negative extraction protocol against several gram positive strains; it was unsuccessful at producing DNA. Therefore, the first step of this summer's project will be to develop a simple extraction protocol for gram positives.

From there, I will test my gram negative nucleotide primers against the gram positive bacterial DNA to determine their viability for use with both types of bacteria. If they are proven to be unsuccessful at amplification, I will return to analysis of the 16s gene maps of all of my target species in an attempt to design new, universal PCR primers.

That is the primary focus of my summer project. I will keep you updated as to my progress.

Until next week, when I can begin posting data and photographs relevant to my project, please enjoy this pic of my dog pack. :)