Essay On Adaptor Ligated DNA

The following tests according to the lab manual were performed: gram stain, fermentation tubes, methyl red, vogues proskauer, sulfur, indole, motility and growing it up on MacConkey agar. The gram stain was performed incorrectly the first time. This is because the decolorizer was not on the bacterium slide for long enough, giving a false outcome.

[1] – Molecular Cell Biology, 7th edition 2012, Harvey Lodish, Chris A. Kaiser, Anthony Bretscher, et al. Macmillian Higher Education.

20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.

Polymerase Chain Reaction was run and the results were placed into an electrophoresis gel to visualize the PCR product. To determine the alleles in the gene, a digest enzyme, Fnu4HI, was placed into the DNA. Tasters would view a cleavage formed at a restriction site in at least one of the alleles, whereas non-tasters would not.

2) Record the shape of the bacteria, the arrangement of the bacteria, and the gram staining characteristics.

Requirments: The total reaction volume was 10 μL, containing 1 μL of 10× LightCycler DNA Master Hybridization enzyme mixture (Roche Diagnostics), 3 mM MgCl2, 0.1 μM hybridization probes, and ARMS primers.

The DNA extraction results, along with the PCR product, did not fare well. There was not enough product produced to be viable in the later stages of the experiment, so a backup was used in place of the original product.

The Basic Local Alignment Search Tool (BLAST) was the final portion of this lab report. BLAST is provided courtesy of the National Center for Biotechnology Information (NCBI). After running the five chosen tests, students were given the two 16S rRNA sequences that were associated with their assigned culture. The BLAST results served to confirm or disprove the hypothesis of what the unknown may be. The BLAST program compared the given 16S rRNA sequence to a database of known sequences and searched for similarity. (10) This search tool is capable of comparing nucleotide or protein sequences to find statistical significance between the matches. A perfect match to the unknown sequence is indicated by a

Before starting all the biochemical tests, an inoculation procedure must happen. An unknown test tube was handed out which contained two different cultures. The unknown culture was place on a nutrient agar plate using the three-streak inoculation technique for isolation (Lab Manual, Chess 2015). After a few days of growing, the presence of two unknown bacteria had a clear visible of separation. Following the isolation streak technique, the gram-staining experiment was performed. The two bacteria were determined: one was Gram-negative bacteria and the other was Gram-positive bacteria.

The results of the traditional mini-prep suggest RNA contamination (> 1.8) and a subsequent above average yield (70-84ug instead of ~1-3ug). The higher than expected yield can be explained since the purity suggested RNA contamination. The kit mini-prep resulted in close to pure DNA except for sample #3 which resulted in a protein contamination (< 1.8). The yields of the mini-prep were within expectations. The results from this experiment were necessary to determine whether the DNA isolated was pB325 plasmid.

Because human DNA is being used, participants must fill out a consent form. Gel electrophoresis will determine the success of polymerase chain reactions. Some of the DNA will be shipped to Cornell University for the Biotechnology Resource Center to sequence. Using the NCBI's BLAST search engine, sequences will be compared to those in NCBI's database to see if the right region was amplified. DNA sequence analysis will help identify SNPs and amino acid present in

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

Both cytosine at CpG sites and cytosine at non-CpG sites can be methylated although with different frequency. It encodes 37 genes, out of which 13 for proteins (phosphorylation enzymes), 22 for tRNA and 2 for rRNA (12S and 16S). The mtDNA also contains a short non-coding region with control elements. It includes an origin of replication and transcriptional promoters. It has two strand promoter, both the heavy strand and the light strand promoter generate proteins. These proteins essentially span the entire genome. It exclude the major non-coding region. Mitochondrial DNA resides inside the mitochondria in a packed form, which is known as mitochromosomes or nucleoids. The transcription factor A (TFAM) is the most abundant component of nucleoids, it is significantly contributes to compact and pack mtDNA, which acts similar to the histone protein in nuclear genome. As similar to the nuclear genome, DNMTs helps in the methylation process of mitochondrial DNA. Depending upon the different level of protein occupancy, DNMTs has different accessibility of mitochondrial DNA in the mitochondrial genome. Another major

Illumina sequencing is an example of next-generation sequencing method. It uses fluorescence-based sequence-monitoring technology and contributes to about 90% of current sequencing data2. In Illumina platform, large numbers of short fragments are sequenced in a single stroke. To do this, firstly, the testing samples need to be cut into short fragments and the section length is depending on the particular sequencing machine model. Illumina sequencing usually use 100-150bp long reads. Longer nucleotide pieces are annealed to a slide using generic adaptors. PCR is then conducted for read amplification, creating a pool of DNA pieces with the same read. Single strands were obtained by separating these copies and were used for sequencing3.

This essay based on the principle of real time PCR which uses CYBR green dye that combines to any double stand DNA. This process included two maim steps. The first step was designing of HSV-1 primer and /or HSV-2 primer (we have chosen only HSV-1). BioEdit software has been used to edit the nuclide sequences where necessary. After that the primer has been ordered. The second step was in the laboratory which included applying the HSV-1 primers to real time PCR protocol. The final results of this protocols aim to demonstrate the quality of HSV-1 primer that we have designed by interpretation three criteria. These are specificity, efficiency and