Biology 475

Molecular Biology

Lab One - Genomic Isolation and PCR

Kelly L. Shipley

Copyright 2003

Introduction Our goal for this lab is to lyse, stabilize and clean genomic DNA samples from bacterial microorganisms for use in PCR. After preparing our cleaned sample DNA, we mix it with all necessary requirements for replication, such as buffers, templates, primers and thermal stable polymerase; it is placed in the PCR machine for cycling 16s product target.

	Genomic Methods and Results
Methods: After isolating and precipitating DNA with sodium acetate and ethanol, we centrifuged our test tubes to pull the DNA into a pellet. The centrifuge was set at 14K rpm for 15 minutes.
Results: Our goal to clean DNA from our samples was hindered by the inability to pull off the top aqueous layer of the phenol/chloroform solution containing DNA without pulling off some of the phenol. Without knowing phenol was present in the new tube, we continued with the procedure and centrifuged. This produced a phenol bubble (see picture) at the bottom of the tube, meaning our DNA pellet had been displaced and our samples were unusable.

	PCR Methods and Results
Methods: These small test tubes are loaded with buffers C, E, J, I, K a set for each of the two types of primer sets, one 16s rRNA and one Red. These are labeled here as 16 and R. The primers are added as part of a cocktail which includes water, forward primer, reverse primer, Taq polymerase and DNA. These test tubes are ready to be placed in the PCR machine.
Results: PCR showed no products at all according to the gel to the right. Each lane is marked above the gel. Lane contents below. Lane 1 holds the DNA marker. Lanes 2 -6 hold buffers C, E, G, I, K respectively with 16s primer. Lanes 7 – 11 hold buffers C, E, G, I, K respectively with Red primer. Lane 12 is empty.	1 2 3 4 5 6 7 8 9 10 11 12

Discussion

Red filamentous microorganisms abound within the bacterial communities of Yellowstone National Park. Our goal to clean and isolate genomic DNA from this bacterium for study requires many different procedures, from the most simple to the most delicate. Our lab experience today demonstrated some of both.

The 16s rRNA gene is specifically the chromosomal target gene sequence. To isolate the chromosomal DNA from the bacterial mat we used phenol/chloroform extraction, hoping to pull off the DNA from the top layer, while the rest of the cell debris lay in the bottom layer. Unfortunately while performing this extraction, I pulled off a small amount of phenol from the tube unknowingly. After centrifugation, instead of a pellet of DNA, I saw a bubble of phenol. My DNA had been displaced and the sample was unusable.

After receiving a previously cleaned sample of DNA, it was added to a primer cocktail for use in PCR. Polymerase Chain Reaction is used to amplify replication of an isolated DNA sequence for future study. For this rapid rate of replication, the cocktail is made of reverse primers, forward primers, buffer, water and our sample DNA along with Taq polymerase. The Taq pol is able to remain stable at the higher temperature, making replication possible. Without any one of these, the rapid amplification will not take place. Due to human error of my own, the cocktail was not mixed correctly or an “ingredient” was omitted completely, my PCR results show absolutely no amplification. There are no bands visible on the PCR reading, meaning no amplification of the target DNA took place. There are several reasons why this may have occurred, all procedural. The cocktail may not have been mixed properly with all necessary ingredients, mixing of the cocktail with the DNA may not have happened, or have been extensive enough as well as the preparation of the DNA not being completed properly. Unfortunately because of any one of these, results are nonexistent for PCR.