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Wolbachia - Lab 3 PCR Flashcards

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1
Q

Summarize Lab 2

A

DNA extraction and purifying all types DNA

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2
Q

What does PCR stand for

A

Polymerase Chain Reaction

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3
Q

What does PCR do?

A

Amplify DNA segments to have enough for arthropod identification

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4
Q

What are the two genes that PCR targets, which one is for Wolbachia, and which one is for the arthropod?

A

16s rRNA - Wolbachia Gene

Arthropod CO1 - Arthropods

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5
Q

What does Arthropod CO1 stand for?

A

Cytochrome C oxidase 1

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6
Q

Where is CO1 found?

A

In the mitochondria. The mitochondia has several copies of mitochondrial DNA.

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7
Q

Why is the CO1 gene excellent for DNA amplifying?

A

Unique to each species so it helps when identifying arthropods

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8
Q

What is 16s rRNA?

A

component of prokaryptic 30s ribosomal subunit

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9
Q

Why is 16s rRNA so easy for PCR to detect?

A

Each animal will have a unique DNA sequence here.

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10
Q

What is PCR Assay and what is the difference between PCR Assay and regular PCR?

A

PCR assay specifically targets Wolbachia. It amplifies DNA in real-time whereas regular PCR amplifies DNA after the reaction is over.

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11
Q

What happens if the 16s rRNA gene is there and if it’s not?

A

If it’s there, the DNA will amplify but if not, nothing will happen.

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12
Q

What is the 16s rRNA DNA sequence used for?

A

To see what strain of Wolbachia an arthropod might have and the relationship between closely related Wolbachia groups.

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13
Q

What is DNA barcoding?

A

using PCR to take the unique DNA sequence and identify organisms

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14
Q

What are the two key components of barcoding?

A

Conserved region and Variable region

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15
Q

What is the conserved region in barcoding?

A

It’s the sequence of DNA that’s the same across a group of organisms. Basically, it’s a sequence in DNA that has remained the same throughout evolution.

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16
Q

What do the two sets of primers do in barcoding?

A

One set will amplify DNA from the arthropods and the other will amplify DNA from Wolbachia.

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17
Q

What is the variable region?

A

A segment of unique DNA, sequenced to classify species (what kind of arthropods).

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18
Q

How many steps are there in PCR and what are they?

A

3 - denaturing, annealing, and extension

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18
Q

True or False, the cycle happens once and then never again?

A

False, the cycle is repeated multiple times, doubling the amount of DNA from the previous cycle.

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18
Q

What is denaturing?

A

The first step of PCR. In this step, DNA is heated to a high temperature.

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18
Q

Why is DNA heated to a high temperature during denaturing?

A

To separate and unwind the double helix to turn the DNA into independent strands.

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19
Q

What is annealing?

A

The second step of PCR. The temperature of the DNA is lowered to bind primers to the denatured DNA strands. The temperature may vary depending on the DNA sequence and the length of primers. There are two types of primers, forward and reverse primers.

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20
Q

What are forward and reverse primers and why do we have them?

A

Primers are small strands of nucleotides that are designed ti target and amplify specific DNA segments. They bind to each end of the DNA segment which is why you need a forward and reverse. They bind depending on the base pair rules, A&T and G&C.

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21
Q

Why is the temperature lowered during annealing?

A

Because it helps the primers bind to the DNA strands.

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22
True or false, nucleotides aren't added during annealing?
True.
23
What is extension?
The temperature is slightly raised so the enzyme Taq polymerase can add the complementary DNA nucleotides to the DNA strands that the primers bind to. It created another identical DNA molecule.
24
Why is the temperature raised during extension?
So the enzyme Taq Polymerase can add the complimentary DNA Nucleotides.
25
Purpose of DNA in PCR?
to get amplified so it can be used to identify arthropods.
25
Why is Primer necessary?
Because DNA polymerase, the enzyme that adds nucleotides to the single DNA strand, can only be added to existing nucleotides.
26
Purpose of nucleotides in PCR
to be added to the single DNA strands to create a new and identical DNA molecule.
26
Purpose of Primer in PCR?
Primers are small lengths of DNA, roughly 20 nucleotides, that bind and amplify specific DNA regions.
27
What is Buffer?
It's added to the PCR mix to maintain pH levels and promotes primer bidning.
28
What is Taq polymerase?
It acts as DNA polymerase to add new DNA bases to the end of the primer sequences.
29
Why is Taq polymerase good for PCR?
It's able to withstand high temperatures to denatre DNA.
30
How many sets of primers are there and what are the primers?
2 sets 1. Wolbachia_F and Wolbachia_R 2. Arthropod_F and Arthropod_R
31
True or false, primers don't have to come in sets but sometimes they will?
False - Primers always come in sets.
32
Why do Primers come in sets?
to direct DNA elongation towards opposite primers at opposite ends of the DNA sequence getting amplified. Basically the forward and reverse primer rule: to go to opposite ends of the DNA strand.
33
what direction does primer add nucleotides in?
5' to 3'
34
What's a PCR mix?
When different ingredients are added in different steps to perform PCR
35
How many types of PCR mixes are there and what are they?
2 1. Taq master mix 2. PCR cocktail
36
What is the Taq master mix?
Supllied directly from a vendor, the Taq maste mix has Taq polymerase, dNTPs (nucleotides), and buffer
37
What is a PCR cocktail?
It has Taq master mix, forward primer, reverse primer, and water
38
Why is water added to a PCR cocktail?
To ensure optimal reaction conditions, prvent degration of nucleic acids, and avoid non-specific amplifying of an unintended DNA sequence.
39
When should you use a PCR cocktail?
When you want to be able to adjust certain measurements based on your experiment or if you're troubleshooting something in your PCR and need to fix something.
40
When should you use a Taq master mix?
When you're doing a normal PCR procedure or need specific and lab measured measurements.
41
For this experiment, is PCR cocktail or taq master mix better?
PCR cocktail
42
What are controls?
They're there to minimize all variables except for the independent variable.
43
What are the two kinds of controls?
Positive and negative.
44
What is a positive control?
A well-understood variable an expected outcome. This doesn't have to be your hypothesis but it just shows that you're experiment is going as expected.
45
What is a negative control?
A sample that shouldn't give a result at all to show that your real samples aren't contaminated. Nothing should happen to them but if something happens, it shows your samples are contaminated.
45
What is the positive control in this experiment and why?
a Wolbachia infected arthropod that amplifies both the CO1 and 16s rRNA gene. This is because it shows that the experiment is working.
45
What's the negative control in the procedure and why?
Any sample with no DNA or just water that you use to show that nothing will happen to it. This is the negative control because it's just used to show there's no contamination.
45
Why should you thaw primers, water, and the master mix before using them?
The materials could settle or degrade during storage so this helps get more accurate and consistent results.
46
What is a thermal/thermo cyler?
It performs PCR by controlling the temperature and temperature changes during PCR.
47
What's a vortex?
machine that mixes small amounts of liquid quickly
48
what's a centrifuge/ mini centrifuge?
a machine that separates fluids by density by spinning liquids to the bottom of the tube. In the mini, there are two different spinning holders to hold different-sized tubes.
49
what are Gloves for?
Protect people and samples from contamination
50
what's the ethanol for?
clean surface before and after the experiment.
51
Why 70% ethanol and not something else?
higher concentrations cause ethanol to evaporate too quickly and can be harsh on certain surfaces. A lower concentration isn't used because it's not as effective.
52
What is the 0.2 ml tube use for?
It's small and has thin walls, allowing for easy heat diffusion from the thermal cycler and the sample
53
What's the 1.5 ml tube used for?
It's the most common tube size and is used for PCR cocktails.
54
What's a balance tube used for?
It's a 1.5 ml tube that hold 260 uL of water to balance the centrifuge.
55
How many DNA samples did we have and what were they?
2 Samples: 1. arthropod samples 2. Wolbachia samples
55
Why do we use pipettes?
For accurate pouring of liquid from one container to another.
56
What is a reagent?
an essential component to the PCR process.
57
How many reagents are there and what are they?
4: 1. Sterile, nuclease free water 2. Taq master mix 3. Wolbachia_F and Wolbachia_R primers 4. Arthropod_F and Arthropod_R primers
58
What is nuclease?
The enzyme that breaks down nucleic acids.
59
Why do we use sterile nuclease-free water instead of other water?
Because tap water or deionized water is not suitable. They may contain nucleases which are DNA-degrading enzymes or other contaminants.

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