Yah Eugene!!

First of all, I wanted to apologize for missing Monday. I hate to miss this lab because I feel like I’m making everyone else pull my weight. I assure you that I would have been of no use on Monday had I came. I’m going to the doctor again today to try to get a handle on these headaches.
The Salzar paper is very interesting. It’s research is very helpful to use in our attempt to discover new species in the family Geodermatophilaceae. I will not focus on its stone sample findings, but rather on the design of PCR primers specific for the family Geodermatophilaceae. I feel that it is also to mention that the paper refers to two probes for specific detection of Geodermatophilus and Modestobacter using fluorescence in situa hybridization. These probes may be useful for us down the road. The Salzar paper designed a forward primer called Geosp2 for positions 156 and 174 and a reverse primer called Geosp1 for positions 725 and 743 on the 16S rRNA gene. specific for the detection of the three genera of the Geodermatophilaceae family. The reason it is specific is because the primers are for conversed only in the Geodermatophilaceae family. They tested the specificity of the primer by comparing its oligonucleotide sequence to all of the sequences available in the GenBank using the FASTA program. The PCR procedure was 40 cycles of 30 s at 93C, 30s at 49C, and 2 min at 72 C, followed by 72C. They compared the amplified products by observing the melting temperature which showed that all of the clones had similar melting temperature.
The sequence of the primers should complete hybridization with species of Geodermatophilus. However, one mismatch was found between one of the primers and Modestobacter multisepatus.Three mismatches were found between the two Blastococcus species and the reverse primer, and one mismatches were found between the Blastococcus species and the forward primer. This is not of great concern because in other families there are many more mismatches than observed between the primers and the Geodermatophilaceae family.
` On Monday, the 16s rRNA gene sequence of a few of our strains was put into BLAST and EZTaxon to compare its relatedness to other species within the Geodermatophilaceae family. The data from this test enabled us to choose two species from our 26 strains to examine using microscopy. We chose the strains K465 (Geodermatophilus) and NC66 (B. jejuensis).
As reported from Sean Michael, on Monday we also made a 1% agarose gel and ran the DNA from the last class. The gel was electrophoresed for about 20 minutes at about 75 volts. The gel was ran twice but neither time produced good results. We also made 10ul spots of the unirradiated control and the irradiated at 9 kGY on the media at which it grew best. 6 strains were put on each plate.
Sean Michael redid the test which had strains that were contaminated. The data from these tests will be added to the master spread sheet.
On Wednesday, we prepared the samples for PCR. A PCR reaction mixture was made. 49.5 ul of the reaction mixture was added to the tubes for adding 0.5 ul of DNA. The DNA is spon down in the centrifuge before being added to the reaction mixture. We did not have enough reaction mixture to run GB-16. Our blank is labeled with a B. The blank will allow us to see if there were any contaminants in our reaction mixture that may have affected our PCR results. The most important step in PCR to avoid primer mismatch is to make sure that the annealing temperature is as high as possible. The higher the annealing temperature the more specific the primer/DNA hybridization will be. The annealing temperature for the forward primer is 50C and the annealing temperature for the reverse primer is 60-62 C. The M is the reverse primer means that a A,C, or G can be present.
We restreaked all our stock plates once. We restreaked KR65 and NC66 twice to be used for microscopy.