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molecular genetics > exam 2 > Flashcards

exam 2 Flashcards

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1
Q

What is the purpose of mechanical grinding in plant DNA extraction?

A

Breaking the cell walls

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2
Q

What is the function of using a detergent in DNA extraction? What is a common one used?

A

To disrupt the cell membranes. Most common is SDS (sodium dodecyl sulfate)

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3
Q

What is the function of EDTA in DNA extraction?

A

Chelates heavy metals like Mg to remove metal cofactors from proteases and nucleases which would degrade DNA. Protect from endogenous nucleases

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4
Q

Why do we limit time in DNA extraction?

A

To prevent degradation

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5
Q

What is the purpose of a buffering agent in DNA extraction? Why is it important in plant DNA extraction? What is an example?

A

Maintains pH since plant vacuole is large and acidic. Example is Tris

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6
Q

What is the purpose of beads in DNA isolation?

A

Grind the tissue of your sample

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7
Q

What are the main components in a PCR reaction?

A

Master mix, forward/reverse primer, nuclease free water, DNA template/control group

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8
Q

What is included in a master mix (4)?

A

dNTPs, Mg2+ ions, buffer, taq polymerase

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9
Q

What are the components of a traditional PCR reaction (7)?

A

Template DNA, Taq polymerase, dNTPs, forward primer, reverse primer, buffer (Mg2+, Tris buffer), water

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10
Q

How long is the extension step? At what temp?

A

3-6 minutes at 72ºC

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11
Q

How long is the denaturation step? At what temperature?

A

2-3 minutes at 95ºC

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12
Q

How long is the annealing step? At what temperature?

A

30 seconds, 45-65ºC

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13
Q

What happens in the repeated cycles during PCR?

A

95ºC- 30 sec- denaturation
45-65ºC- 30 sec- annealing
72ºC- time varies- extension

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14
Q

How do you determine how long the extension step should be based on kB length?

A

1 minute per kB

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15
Q

What precipitates out RNA in plant DNA isolation?

A

LiCl

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16
Q

What solubilizes membranes in plant DNA isolation?

A

SDS

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17
Q

What controls the pH of the extraction in plant DNA isolation?

A

Tris-Hcl

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18
Q

What binds divalent metal ions like Mg++, inactivating nucleases in plant DNA isolation?

A

EDTA

19
Q

What precipitates DNA out of the solution of cell contents in plant DNA isolation?

A

Isoproponal

20
Q

What removes cell debris in plant DNA isolation?

A

Centrifugation

21
Q

What binds to divalent metal ions, like Mg++ to inactivate nucleases in human mtDNA isolation?

A

Chelex

22
Q

What denatures protein and RNA in human mtDNA isolation?

A

High heat

23
Q

What maintains the pH of the extraction in human mtDNA isolation?

A

Tris-HCl

24
Q

What removes cell debris in human mtDNA isolation?

A

Centrifugation

25
Q

What components of plants can affect purification (2)?

A

Contaminants or secondary metabolites

26
Q

What are some considerations when it comes to purifying DNA (4)?

A

-neutralizing and avoiding DNA degradation and DNA degrading enzymes
-removing DNA from the natural barriers of the cell
-purifying DNA away from other nucleic acids such as RNA
-purifying DNA away from other contaminants or secondary metabolites

27
Q

Which direction does DNA synthesis occur in?

A

5’ to 3’

28
Q

How can you determine SNP variability?

A

Sequencing data from PCR product

29
Q

Can agarose gel electrophoresis indicate DNA sequence differences?

A

No, only band size

30
Q

What does “cleaning up” the PCR product mean?

A

Removing residual template DNA, proteins, or primers

31
Q

What happens if you don’t clean up the DNA?

A

Contaminates PCR product and inhibits sequencing reaction

32
Q

What concentration of DNA is sent for sequencing?

A

100ng

33
Q

What is included in sequencing reactions?

A

DNA, water, and primer

34
Q

What volumes change when sequencing?

A

Water and sample volumes, company dependent

35
Q

How many primers will be included in one tube to be sent to sequence?

A

One, either forward or reverse

36
Q

What does DNA polymerase require for elongation? What is its purpose?

A

3’-OH. Catalyzes the formation of a new phosphodiester bond.

37
Q

What provides the 3’-OH for DNA polymerase?

A

Primers

38
Q

What is a positive control?

A

Confirms that your assay or experiment worked. Gives a parallel comparison.

39
Q

What is a negative control?

A

Confirms you are not getting false positive results. Demonstrates what a negative result looks like

40
Q

What are the two most variable components in a PCR reaction?

A

DNA template/ control group and primers

41
Q

Where is DNA in the clean up protocol?

A

Spin column

42
Q

What volume of membrane binding solution is used to clean PCR amplification?

A

Equal volume of PCR amplification. 20uL of PCR = 20uL of membrane binding solution

43
Q

What is elution in PCR purification?

A

DNA purification from the spin column. Done with water and centrifuge.

molecular genetics (5 decks)

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