In this virtual lab you will assume the role of a lab technician in a modern molecular biology laboratory. As such, you are responsible for providing lab results to medical doctors for use in diagnosing their patients. Be sure to follow the steps of the procedure in order and to make use of the notes on the right side of the computer screen. As you work through the lab, answer the following questions:
1. As the medical technician in charge of this investigation, what are you trying to determine about the tissue sample provided to you?
A-About the tissue sample is to identify a bacterial sample received from a clinician.
2. How did you prepare the DNA to be used in this investigation?
A-Well first the DNA consists of dissolving the cell wall with a digestive buffer, and what the buffer contains proteolytic enzymes that technically eat the cell wall. This will take about several hours. Then the DNA is contained in supernantant (the liquid) then transfred to the PCR tube.
3. Describe how PCR is used to make copies of DNA sequences. Use the animation and notebook entries in the PCR Amplification step to guide your answer. Note that you may replay the animation as needed.
A-PCR is used to make copies of DNA sequences by the fact you first start off by adding PCR Master Mix solution to the sample of DNA. You then prepare negative and positive control reactions. It is then put into a machine, and it determines the temp, the time, the cycle number, melt, anneal, and extend. The first step is to melt so it can separate the two DNA chains in the double helix by heating the vial containing the PCR reaction mixture to 95 degrees celcus for 30 seconds. Then the vial is cooled to 60 degrees celcus. At that temperature the primers will bind (anneal) to the single strand of DNA. The last step (extend) is to let the DNA extend the copy of DNA strand by rising the temp. to 70 degrees celcus for 45 seconds. When you do these three steps each is carried out in the same vial, then at the end of a cycle, each piece of DNA in the vial has been duplicated.
4. Summarize the technique used to purify the PCR product.
A-The technique that is used to purify the PCR product is the PCR Master Mix solution. What it is, water; a buffer to keep the mixture at the correct pH for the PCR reaction; large quantities of the four nucleotides adenine, cytosine, guanine, and thymine; large quantities of oligonucleotide DNA primers that bind the 16S rDNA region to indicate the replication process. What it does is basicly you mix the product with DNA mix it in the machine let it set and it makes copies of DNA.
5. What is produced during the sequencing prep PCR run? Use the animation and notebook as needed in thinking through your answer.
A-The is produced during the sequencing prep PCR run is when the DNA replication starts. You have to melt, anneal, and extend.
6. Describe how the automatic sequencer determines the sequences of the PCR products.
A-When it is in the machine it determines the temperature, the time remaining, cycle number, melt, anneal, and extend.
7. What does BLAST stand for?
A-BLAST stands for Basic Local Alignment Search Tool
8. What conclusions did you make using the results of the BLAST search? Did these conclusions support a clinical diagnosis for the patient (what disease did they have)?
A-What BLAST does is it will start with the reference of the search program, which is the number of letters in the sequence and then the number of letters in the database. Then a graphic representation of the sequence matches, and a list of matches. There are a lot of sequences in the database and some of them are from the same species and therefore might be similar.
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