What is the piece of DNA used for identifying bacteria, and what does this indentification rely on? - ANS -The region that codes for a small subunit of the ribosomal RNA (16S RNA)
the identification relies on matching t
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What is the piece of DNA used for identifying bacteria, and what does this indentification rely on? - ANS -The region that codes for a small subunit of the ribosomal RNA (16S RNA)
the identification relies on matching the sequence from your sample against a database of all known 16S rDNA sequences
Describe the process of extracting bacterial DNA (sample preparation) - ANS -1. dissolve the cell wall with digestive buffer
2. heat sample in a water bath 100°C to denature proteolytic enzymes from digestive buffer
3. spin sample in centrifuge
4. transfer supernatant (the liquid) to PCR tube
Why do you heat the sample after adding the digestive buffer? - ANS -The buffer contains proteolytic enzymes that dissolve the cell wall so DNA can be extracted. Once it does this, we must denature the buffer to prevent the proteolytic enzymes from interfering with the other enzymes used during PCR
Where is the extracted bacterial DNA in the centrifuge tube? - ANS -The cellular debris is spun down in the centrifuge and appears as a solid deposit (pellet). The DNA is contained in the supernatant (the liquid) that is transferred to the PCR tube
How do you dissolve the cell wall to extract the bacterial DNA? - ANS -proteolytic enzymes in digestive buffer dissolve the cell wall
What is PCR - ANS -polymerase chain reaction is a technique that allows many copies of DNA to be made from a small original sample
Describe what happens in PCR - ANS -In normal cells, the dsDNA is unzipped with an enzyme to start the replication process. In PCR, ssDNA is made by heating a chromosome fragment to 95°C. It is then cooled.
Why is the sample cooled after the 95°C water bath? - ANS -so that the primers anneal to the og DNA strands and DNA polymerase can bind and copy each strand
What is used to obtain the desired portion of the DNA in this lab?? - ANS -oligonucleotide primers that specifically bind to regions flanking the 16s rRNA gene. This binding initiates the replication process
Describe the steps of PCR - ANS -1. add PCR Master Mix solution to sample DNA, positive control, and negative control
2. add positive and negative controls to their tubes
3.load tubes into thermocycler (PCR machine), run and then remove
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