LAB 03B: HHMI BACTERIAL
IDENTIFICATION VIRTUAL LAB. EXAM
QUESTIONS AND ANSWERS (VERIFIED
AND UPDATED)
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
@2026/2027 ALLRIGHTS RESERVED.
, 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
4. insert microconcentrator column of appropriate size into collection tubes
5. add 400µL of buffer to columns
6. add entire PCR content (~100µL) to column
7. put positive and negative control tubes on ice
8. Spin column at 3,000 rpm in a fixed-angle centrifuge for 15min
9. Remove column and discard collection tube
10. invert column, attach to new collection tube, add 50µL of buffer to inverted column
11. spin inverted column 3,000rpm for 2min
12. discard column
@2026/2027 ALLRIGHTS RESERVED.
IDENTIFICATION VIRTUAL LAB. EXAM
QUESTIONS AND ANSWERS (VERIFIED
AND UPDATED)
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
@2026/2027 ALLRIGHTS RESERVED.
, 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
4. insert microconcentrator column of appropriate size into collection tubes
5. add 400µL of buffer to columns
6. add entire PCR content (~100µL) to column
7. put positive and negative control tubes on ice
8. Spin column at 3,000 rpm in a fixed-angle centrifuge for 15min
9. Remove column and discard collection tube
10. invert column, attach to new collection tube, add 50µL of buffer to inverted column
11. spin inverted column 3,000rpm for 2min
12. discard column
@2026/2027 ALLRIGHTS RESERVED.
