More DNA......

At the beginning of this week, we ran the DNA we extracted from 15 Gobi samples on agarose gel (we extracted this DNA last week). Unfortunately, when we looked at our gel under fluorescent light, there were no DNA bands to be found (except the ladder). This could be because we performed the extraction protocol incorrectly or because we loaded the gel incorrectly. So, we ran our DNA again…and it failed to show up…again. We also spotted controls and samples that had 9 kGys of radiation (from our 26 strains) onto optimal media. In our lecture, we discussed the 16s rRNA sequence, which has about 1500 nucleotide positions. We work with the 16s sequence because it has variable regions (enough variations to separate species). Primers are made from highly conserved regions (because that way they can work for many organisms). Lastly, we compared some of our strains in EzTaxon and BLAST to see what organisms they are most closely related to.On Wednesday, we restreaked our stock plates and put them in the 25C incubator. We put the old stock plates in the cold room (we restreaked two stock plates for KR-65 and MCC-66 so we could use them to take to the microscopy lab). We also set up PCR using the DNAs we had extracted so far (meaning 21 separate DNAs). We used a premix that Dr. Rainey so kindly made for us. In lecture, we talked about PCR in depth. We learned that the critical part in PCR is annealing temperature which determines whether or not the primer will bind to the DNA. We also learned that we must mix our DNA with buffer, primer 1 and 2, taq polymerase, dNTPs, and water (we made a 50 uL reaction using 49.5 uL of premix—which is everything listed except the DNA---and .5 uL of DNA). We also had to make a control which just contained premix (no DNA). Our group was not able to use our GB16 strain because we rain out of premix (we spilled some, accidentally..oops!). All in all, it was a good, productive week. We will all be sad when Dr. Rainey is not in class on Monday.