.....impt of staying awake in BIOL 101...........

So this week went well! On Monday we received our test grades which were fair! The tasks which were performed included: making agarose gel, irradiated our organisms at 9kGy, and discussed gene sequencing. First, we started with making agarose gel and running our DNA. Actually, Rainey made the agarose gel and my lab partner ran the DNA. Our DNA results didn’t go as planned. The agarose gel broke and the experiment didn’t go as planned. There was no sign of any DNA present. Next was the irradiation. For this we used a control (no irradiation) and organisms which were irradiated at 9kGy. We spotted six spots per plate (3 controls and 3 irradiated organisms). This was a continuation of a previous experiment. For the previous experiment we irradiated organisms at 0, 3, 6, and 16kGy. There were colonies present at 0, 3, and 6kGY but none at 16kGy, so this experiment was performed to see if there would be any growth between 6 and 16kGy. Next, there was the DNA sequencing discussion. This was very informative! Of some things that I learned were the 3 ribosomal genes which are: 16S, 23S and 55S. The reason that 16S is so often used is because at this level there are variable regions and also highly conserved regions. These highly conserved regions are found in bacteria and certain species so 16S is specific. Also, we have a 16S description for every described species, so there is more information available for 16S sequencing. I also learned that a partial 16S sequence was 650-900 base pairs where as a full 16S sequence was 1900 base pairs. This knowledge was beneficial because next we used online databases such as eztaxon and BLAST to sequence some of our strains. Most results from these databases turned up many results which varied in there percentages of similarities. WE used the result with the highest percentage to classify our organism.
On Wednesday we streaked new stock culture plates, analyzed our old stock culture plates, and set up PCR using all DNA extracted thus far. Streaking our new stock culture plates was nothing new; however, we got a chance to find out which ones were contaminated which may have led to some of our other contaminations (in my opinion). Next we set up the PCR experiment. From Monday and Wednesday discussions I learned quite a few things about PCR primers. PCR binds to DNA and amplify it. The most important role of primers are annealing. The annealing temperature of primers are: for every G+C 4 degrees and for every A+T 2 degrees. Sorry back to what we did! We used 18 strains to sequence and one control. Our PCR premix was 50 micro liters for each. It included 95 micro liters of buffer, 95 micro liters of dNTPs, 9.5 microliters of primer 1 and 2, 731.5 microliters of water and 4.75 micro liters of Taq, and .5 micorliters of DNA. These calculations were pretty exact; however, we still managed to NOT have enough premix. This was disappointing because we were not able to perform the experiment on all of our strains; however, I did learn how to correctly use the pipette FINALLY!! After preparing the premix we centrifuged our tubes, then add the DNA. We were supposed to start our sequencing, but time did not allow ((so sad))!! That was our week in a nutshell J