Polymerase chain reaction and 16S

This week, we learned a lot about the 16S RNA sequence and about running a PCR. The 16S RNA sequence contains about 1500 Nucleotide bases. These fairly large sequences contain highly conserved regions that show their relationship to specific genera and families, but there is enough variation to distinguish between species. Whereas the 5S is too highly conserved and too small to differentiate between species. The PCR technique uses forward and reverse primers to bind to conserved regions and amplifies the 16S gene. A good replicated gene sequences contains about 600 to 900 bases.
On Monday, we spotted 10µl of a control and the 9kGy treated stains on the media they grew best on. Then we ran a 1% agrose gel on fifteen DNA extractions from March 3. We had to run the gel twice and neither gel showed a strong presence for DNA. Either something went wrong during the DNA extraction or the wells weren’t loaded properly. After, we used to EzTaxon and Blast to find which species the strains that were previously sequenced were most closely related to. Between the three groups, we had at least one stain from each genus.
On Wednesday, we re-streaked new stalk plates and streaked six stains (at least one from each genus) twice which we will use for microscopy next Monday. Then we prepared to run a PCR on the extracted DNA from the class up to this point. Our group has 21 different DNAs. Dr. Rainey mixed the correct amounts 10XBuffer, dNTPs, Primer I, Primer II, water and Taq (a heat-stable DNA polymerase) to make the pre-mix. We added 49.5 µl of the pre-mix and 0.5 µl of DNA to the PCR tubes. One tube contained only the premix which was used for the negative control. The tubes were placed into the PCR machine. The PCR machine contains a heated lid to prevent condensation forming on the lids of each tube. The machine was started and if went through 40 cycles of heating and cooling. It heats up to melt the DNA and separate the stands, then it cools down to the annealing temperature to allow the primer to bind to the DNA. The annealing temperature is found by adding 2ºC for every A-T pair and 4ºC for every G-C pair in the primer. We found the annealing temp to be 50ºC for primer I and 60 to 62ºC for primer II. After the forty cycles of temperature changing, the machine maintains a temperature of 72ºC for 10 min to make sure all the genes are elongated. Then process is complete and the machine keeps the DNA at 4ºC until you take them out.