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!