Laboratory #11 – PCR, Gel Electrophoresis, & Sequencing

These flashcards cover key concepts and procedures related to the Amplification of 16S rRNA gene, PCR methodology, Gel Electrophoresis techniques, and the final sequencing analysis.

What is the objective of Laboratory #11?

To successfully amplify the 16S rRNA gene of unknown bacteria, confirm on gel electrophoresis, and obtain the DNA sequence for identification.

What are the two powerful tools used to identify an unknown microbe in microbiology and molecular biology?

Polymerase Chain Reaction (PCR) and Gel Electrophoresis.

What is the significance of the 16S rRNA gene in bacteria?

It is evolutionarily conserved and serves as a 'zip code' for the genetic identity of bacteria.

What are the three defined steps of the polymerase chain reaction?

Denaturation, Annealing, and Extension.

What is the purpose of gel electrophoresis in the PCR process?

To separate PCR DNA on the basis of size and charge through a gel matrix.

How can DNA be visualized during gel electrophoresis?

Using an intercalating dye that fluoresces under blue light.

What are the materials used in the PCR process?

Unknown sample, P20 pipetter, PCR tube, sterile sticks, Taq 2X Master mix, primers, and nuclease free water.

What is the first step in the PCR procedure?

Take 1 colony from the soil streak plate and resuspend it in 100ul of dH2O.

What reagents are added to the PCR tube?

10ul of Taq 2X Master Mix, 10ul of Forward Primer, 10ul of Reverse Primer, 10ul of nuclease free water, and 10ul of bacterial unknown sample.

What is done with the PCR tube after adding reagents?

Place the PCR tube in the thermocycler.

What is the first step of the gel electrophoresis procedure?

Combine 5ul of loading dye to your PCR reaction.

How much of the PCR reaction is loaded into a gel well?

20ul of the PCR reaction.

What should you write on the diagram while loading the gel?

Your name and unknown #.

What tool is used for sequence identification after PCR?

NCBI Blast.

What website is used for NCBI Blast analysis?

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGETYPE=BlastSearch&LINKLOC=blasthome

What is the role of Taq 2X Master mix in PCR?

It contains the necessary components for the PCR process, including DNA polymerase.

What does PCR recreate in a tube?

DNA amplification.

What is the final outcome of sequencing the 16S rRNA gene?

It provides the exact sequence of the microbe's DNA.

After confirming the PCR reaction, what is the next step?

Send the sample for sequencing.

What is an intercalating dye used for in gel electrophoresis?

To visualize DNA under UV light.

What is the purpose of the sterile sticks in the materials list?

To safely transfer bacterial colonies.

Why is the 16S rRNA gene a good target for amplification?

Because it is conserved across different bacterial species.

What is the significance of comparing sequences to a national database?

To determine the bacterium's identification at the genus level.

During gel electrophoresis, how is DNA separated?

Based on size (base pairs) and charge.

What happens during the Denaturation step of PCR?

The DNA double strand is separated into two single strands.

What occurs during the Annealing step of PCR?

Primers bind to the template DNA strands.

What is the function of the primers in PCR?

They provide a starting point for DNA synthesis.

What is the role of the thermocycler in PCR?

It controls the temperature cycles necessary for DNA amplification.

What does the term 'nuclease free water' refer to?

Water that is free of enzymes that could degrade DNA.

What is the composition of the PCR reaction mixture?

Taq Master mix, primers, water and the bacterial sample.

Why is it important to streak the unknown sample on a nutrient agar plate?

To isolate individual bacterial colonies for further study.

What does BLAST stand for in the context of bioinformatics?

Basic Local Alignment Search Tool.

How does gel electrophoresis help confirm a successful PCR reaction?

By visualizing the amplified DNA fragments on the gel.

What effect does the electric current have during gel electrophoresis?

It causes the DNA to migrate through the gel towards the positive electrode.

What can be inferred if no bands are visible on the gel after electrophoresis?

The PCR reaction may have failed or the DNA may not have amplified.