Back to the Daily Grind

This week’s class began with a group discussion that covered the importance of using the 16sRNA gene sequence to classify organisms. This ~1500bp sequence is highly conserved region of DNA makes it perfect for our comparisons. We also discussed the 5sRNA and 23sRNA and why those sequences are not used. The 5sRNA at ~100-150bp has too little variation, too small and too conserved to be useful. The 23sRNA at ~3000bp never quite took off because there was such a massive amount of 16sRNA sequenced and to sequence the all those organisms’ 23sRNA would be quite a task.
The importance of the high conserved areas on the gene became quite apparent when the discussion of primers began. The sections “27 forward” and “15-25 reverse” are two conserved areas in the 16sRNA that we can exploit by making primers that can replicate the DNA of our three genera. These primers allow the DNA to be copied in both directions to generate many copies of the 16sRNA gene sequence when using Polymerase Chain Reaction Method. To get the complete sequence if our replication pauses, we can use other primers set to start at other areas in the sequence (fox forward at ~519bp). A good amplification is ~900bp and the average is ~600bp
Our first task involved spot plating 10 microliters of our twenty-six strain suspensions exposed to 9 kGy of radiation. We divided the plates into 6 quadrants because we are measuring a qualitative characteristic of our organisms. Each was plated of their optimal media. The second task involved the production of a gel and buffer solution to run our Little Red Hill DNA extract samples (LRH-1, 2, 4, 5, 9, 11-17). Unfortunately when I tried to remove our first gel it broke into many pieces. I made a second gel and re-ran the electrophoresis. When placed under the UV light, only a few faint bands appeared which could mean there was no DNA or not enough DNA was present (we will test this by utilizing PCR). The last task of the day involved looking up the forward 16sRNA sequences on BLAST and EzTaxon. This provided us with a good idea of the relationship of our strains to our three genera.
The next began with a group discussion on what organisms we will bring down to the Microscopy lab on Monday based on the BLAST and EzTaxon results. The two strains we chose were: AT04-166-14 (strain 40) and AT03-37-7 (strain 37). We then moved on to a discussion about PCR which involved discussing the process to figure out the correct annealing temperature for our strains (the most important step in PCR is the annealing). We then discussed the proper order of making PCR Master Mix: buffer 10 µl, dNTPs 5 µl, primer one 0.5 µl, primer two 0.5 µl, water 38.5 µl, Taq polymerase 0.25 µl and your DNA 0.5 µl. The mix is added to the tube first and then the DNA is added, centrifuged and then placed in the PCR machine. We made the PCR solutions using the DNA we’ve extracted so far in the semester (labeled tubes red and renumbered according this order: strain 18, 27, 29, 34, 44, and 48: LRH-1, 2, 4, 5, 9, 11-17).
Our next task involved making new stock plates of our twenty-six strains. Two of our stock plates were contaminated so we tried to pick around the contamination, if that fails will we have to restreak from the strain vials. We made duplicates of AT04-166-14 (strain 40) and AT03-37-7 (strain 37) so we can bring them to the lab on Monday. The next step involved determining the tests that we will run again because either the sample was contaminated, was missing or had dried out due to poor parafilming technique. On Friday my group is tentatively scheduled to meet and re-do some of these corrupted tests.