exam 2

Question 1

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

Answer 1

Breaking the cell walls

Question 2

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

Answer 2

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

Question 3

What is the function of EDTA in DNA extraction?

Answer 3

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

Question 4

Why do we limit time in DNA extraction?

Answer 4

To prevent degradation

Question 5

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

Answer 5

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

Question 6

What is the purpose of beads in DNA isolation?

Answer 6

Grind the tissue of your sample

Question 7

What are the main components in a PCR reaction?

Answer 7

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

Question 8

What is included in a master mix (4)?

Answer 8

dNTPs, Mg2+ ions, buffer, taq polymerase

Question 9

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

Answer 9

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

Question 10

How long is the extension step? At what temp?

Answer 10

3-6 minutes at 72ºC

Question 11

How long is the denaturation step? At what temperature?

Answer 11

2-3 minutes at 95ºC

Question 12

How long is the annealing step? At what temperature?

Answer 12

30 seconds, 45-65ºC

Question 13

What happens in the repeated cycles during PCR?

Answer 13

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

Question 14

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

Answer 14

1 minute per kB

Question 15

What precipitates out RNA in plant DNA isolation?

Answer 15

LiCl

Question 16

What solubilizes membranes in plant DNA isolation?

Answer 16

SDS

Question 17

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

Answer 17

Tris-Hcl

Question 18

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

Answer 18

EDTA

Question 19

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

Answer 19

Isoproponal

Question 20

What removes cell debris in plant DNA isolation?

Answer 20

Centrifugation

Question 21

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

Answer 21

Chelex

Question 22

What denatures protein and RNA in human mtDNA isolation?

Answer 22

High heat

Question 23

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

Answer 23

Tris-HCl

Question 24

What removes cell debris in human mtDNA isolation?

Answer 24

Centrifugation

Question 25

What components of plants can affect purification (2)?

Answer 25

Contaminants or secondary metabolites

Question 26

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

Answer 26

-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

Question 27

Which direction does DNA synthesis occur in?

Answer 27

5' to 3'

Question 28

How can you determine SNP variability?

Answer 28

Sequencing data from PCR product

Question 29

Can agarose gel electrophoresis indicate DNA sequence differences?

Answer 29

No, only band size

Question 30

What does “cleaning up” the PCR product mean?

Answer 30

Removing residual template DNA, proteins, or primers

Question 31

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

Answer 31

Contaminates PCR product and inhibits sequencing reaction

Question 32

What concentration of DNA is sent for sequencing?

Answer 32

100ng

Question 33

What is included in sequencing reactions?

Answer 33

DNA, water, and primer

Question 34

What volumes change when sequencing?

Answer 34

Water and sample volumes, company dependent

Question 35

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

Answer 35

One, either forward or reverse

Question 36

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

Answer 36

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

Question 37

What provides the 3’-OH for DNA polymerase?

Answer 37

Primers

Question 38

What is a positive control?

Answer 38

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

Question 39

What is a negative control?

Answer 39

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

Question 40

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

Answer 40

DNA template/ control group and primers

Question 41

Where is DNA in the clean up protocol?

Answer 41

Spin column

Question 42

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

Answer 42

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

Question 43

What is elution in PCR purification?

Answer 43

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