The water samples were from the Pacific Ocean, on the waters surrounding the Catalina Islands. The samples were taken from varied depths, ranging from _____ to ____ feet deep. Water samples were collected from SPOT locations. After the samples have been collected, multi-cellular organisms and other suspended materials were filtered out. The remaining materials were then further purified and concentrated by using ultrafilteration. These final samples were then mixed with PEG precipitate and wers stored at –20 degrees Celsius to keep them viable for the duration of the study.
The next step involves the preparation of the material for the extraction of the DNA. This preparatory stage is a multi-step process that involves centrifuging, air drying, and suspending. Samples were centrifuged at 4 Co for 30 minutes. The supernatant is discarded, and then the remaining samples were air-dried for 45 minutes. After air-drying, the samples were then suspended by the addition of 1X TE (500 ul). After the preparation for extraction, the samples are ready for the actual DNA extraction. First, 30 ul of 10% SDS and 3 ul of protease K were mixed.
This mixture was placed in an incubator for 1 hour, with temperature kept at a constant 37 Co. Then 100 ul of 5M NACL was mixed and vortexed. To this, 80 ul of CTAB solution was added. This new mixture was again incubated for 10 min at 65 Co. After this, 500 ul of choloform was added, and the resulting solution is spun at 100k rpm for 2 minutes. The bottom layer is removed, and DNA was isolated by using two rounds of phenol extraction followed by three rounds of phenol/cholorform extraction using the 1:1 ratio.
The sample is again spun at 10k rpm for 2 minutes, and the bottom layer is discarded. To the remaining sample, 500 ul of 100% isopropyl alcohol is added. The DNA was then set to precipitate overnight at –20 Co. After one night, the sample was centrifuged at 14g at 4 Co for 30 minutes. Then, after the addition for 70% ethanol, the supernatant was again discarded, and the resulting pellet was air-dried. Lastly, the pellet was again suspended in this medium. The PCR process consisted of the use of g23 primers. One primer was an MZIA1bis (5’-GATATTTGIGGIGTTCAGCCIATGA-3’).
The other primer was a MZIA6 (5’-CGCGGTTGATTTCCAGCATGATTTC-3’). These are the oligonucleotides used by PCR to amplify approximately 500 bp segment of g23 of the viral samples. From each DNA sample, 1 ul was added to the PCR master mix. The master mix consisted of Taqpolymerase (NEB), . 20mM concentration each of DNTP, 40 pmol of each primer, and 1. 0 unit of the Taq polymerase (NEB). Negative control contained water and the master mix. PCR was performed using denaturation period, annealing period, and a final extention period. Denaturation was performed at 94 Co for 90 seconds.
Then, denaturation continued at 94 Co for 45 seconds for 30 cycles. After this, annealing for 1 minute at 50 Co was done, and again for 45 seconds at 72 Co. Lastly, extention was done at 72 Co for 5 minutes. After the purified DNA has been extracted, gel electrophoresis analysis of PCR- amplified g23 fragments was done, along with the negative control and the ladder. The gel was made of a 1% mix of pure agrose with a buffer mix. PCR products were purified with a gel extraction kit (Qiagen). Then cloning was performed using a cloning kit (Promega).
The white clones which form each library were gathered and streaked onto agar plates. On each agar plate there were around 100-200 colonies and random colonies were sent for sequencing. There was a total of 96 colonies that were sent out for sequencing. Once the sequencing results were obtained, the BLAST program was used to identify specific T4 viruses. Sequences were aligned by the program BIOEDIT, and the results were further aligned manually. Once alignment was finished, the g23 sequences were compared to one another in order to determine viral diversity.
The base pairs from 400-600 showed a conserved region, and this was the part of the DNA that was analyzed. Sequences obtained from this lab were compared with other identified sequences. A phylogentic analysis was done which compared the sequences of the T4 viruses obtained in this experiment with the other known viruses. This phylogenic tree was made using a program called Genious. The main tree that was made became the basis for the analysis of the g23 sequences of different samples.
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