Exam 2

Question 1

One of the first steps of a DNA extraction involves a mortar and a pestle, what is this step?

Answer 1

Grinding

Question 2

A buffer containing enzymes is used to “open the cells” during incubation. This process is known as?

Answer 2

Lysis

Question 3

The amount of enzymes used in a reaction affects the efficiency of the enzymes activity. Name 3 other factors that affect enzyme activity.

Answer 3

Temperature, pH, and time

Question 4

After lysing the cell open, the sample is centrifuged after which the DNA is located where in the tube?

Answer 4

Precipitant

Question 5

When extracting DNA, the precipitate is added to the QiAshredder and centrifuged. What happens to the DNA during this step?

Answer 5

Binds to the column

Question 6

After isolating DNA from the gel, a gel electrophoresis was ran for the purpose of ___ the DNA.

Answer 6

Analyzing

Question 7

The purpose for washing a column containing bound DNA is?

Answer 7

Washing away impurities

Question 8

After the last column wash, the DNA is removed from the column by the process of?

Answer 8

Eluting

Question 9

Name 3 reagents that are needed in a PCR MasterMix for a successful reaction to occur.

Answer 9

GoTaq, forward primer, reverse primer

Question 10

Cells that can easily incorporate DNA from their environment are called __ cells.

Answer 10

Competent

Question 11

In a PCR reaction, what is responsible for replicating the DNA?

Answer 11

Forward and reverse primers

Question 12

Primers consist of ___.

Answer 12

Oligonucleotides

Question 13

Name a DNA polymerase used in a PCR that does not have proofreading abilities.

Answer 13

Taq polymerase

Question 14

There are three primary temperature changes in PCR reaction:

Answer 14

Denaturation at 94C
Annealing at 54C
Elongation at 72C

Question 15

The temperature a PCR product should be stored at is?

Answer 15

4C

Question 16

Name the machine that we used to run our PCR in.

Answer 16

Thermocycler

Question 17

What does PCR stand for?

Answer 17

Polymerase chain reaction

Question 18

Name the process of using electricity to run DNA through a substance such as an agarose matrix.

Answer 18

Gel electrophoresis

Question 19

The PCR products were loaded onto a ___ to determine if the DNA was present or absent.

Answer 19

Agarose gel

Question 20

After amplifying extracted plant DNA by PCR, and after running the PCR produce through a gel; how was the DNA removed from said gel?

Answer 20

Excision

Question 21

A “ladder” is also known as..

Answer 21

Gene ruler or molecular ruler

Question 22

DNA shows up on a gel in the form of a ___.

Answer 22

Band

Question 23

Absorption of DNA to a silica membrane is dependent on ..

Answer 23

Charge and pH

Question 24

What is the optimal pH for absorption of DNA onto a silica membrane?

Answer 24

7.5

Question 25

3M sodium acetate can be added to a sample to ___ pH.

Answer 25

Lower

Question 26

Name the plasmid that was used to clone the RcbL gene.

Answer 26

pGEM-T

Question 27

What characteristic of a pGEM-T vector makes is easy to ligate PCR products onto it?

Answer 27

Overhangs

Question 28

Name the gene on the pGEM-T vector that indicates if the RcbL gene was successfully transformed or not.

Answer 28

LacZ

Question 29

What does the LacZ gene interact with?

Answer 29

B-galactosidase

Question 30

The process of "introducing" ligated vectors into the E. coli is ___.

Answer 30

Transformation

Question 31

Name the technique by which the cell walls were altered during transformation.

Answer 31

P-shock

Question 32

The optimal temperature for "exciting the host cells" during transformation is what?

Answer 32

42C

Question 33

After heat shocking, how was the reaction quenched?

Answer 33

Ice bath

Question 34

Name the bacteria that was used for transformation and name one reason why it was chosen.

Answer 34

E. coli

Question 35

Explain each of the following results: no colonies, blue colonies, and white colonies.

Answer 35

- No colonies -> transformation failed - Some blue colonies, but no whites -> the ligation reaction failed - Less than 50% white colonies -> ligation conditions suboptimal or nuclease contamination of the ligation may have occurred - White colonies -> ligation successful

Question 36

What is T4 DNA ligase used for?

Answer 36

- catalyzes the formation of a phosphodiester bond - repairs single-strand nicks - joins DNA fragments with either cohesive or blunt termini

Question 37

In the protocol for isolation of plasmid DNA it states that after the resuspension, the cells should not be vortexed because of what reason?

Answer 37

Shearing of the DNA

Question 38

Name the medium that was added to the transformed cells for the purposes of repair and recovery.

Answer 38

SOC medium

Question 39

What temperature were the transformed cells incubated at?

Answer 39

65C

Question 40

After isolation, DNA from three organisms was present. Name the three organisms.

Answer 40

E. coli, part of plasmid, rubisco

Question 41

Name the two primers used to amplify the insert DNA from the plasmid.

Answer 41

Sp6 and T7

Question 42

The fragment size for the RbcL gene should be around ___ bp.

Answer 42

599-750

Question 43

If a blue colony was picked, the fragment size should be around ___ bp.

Answer 43

150

Question 44

If two bands were present when the PCR product was analyzed, one at 150bp and the other at 750bp, what would they be?

Answer 44

- 150 would be plasmid | - 750 would be gene+plasmid

Question 45

rbcL

Answer 45

Codes for large subunits of rubisco

Question 46

GoTaq green master mix

Answer 46

``` Taq polymerase MgCl2 dNTPs Loading buffer Green loading dye ```

Question 47

MgCl2

Answer 47

Acts as a cofactor to Taq polymerase reaction, acts as catalyst to reaction

Question 48

dNTPs

Answer 48

Free nucleotides to add to growing fragment

Question 49

Loading buffer

Answer 49

For better amplification of GC and AT rich targets

Question 50

Green loading dye

Answer 50

Allows tracking during gel electrophoresis

Question 51

Buffer AW1

Answer 51

Binding buffer

Question 52

Buffer AW2

Answer 52

Wash buffer

Question 53

Buffer AE

Answer 53

Elution buffer

Question 54

Buffer AP1

Answer 54

Lysis buffer

Question 55

Buffer P3

Answer 55

Neutralization buffer for plasmid preparation

Question 56

PCR with primers Two steps

Answer 56

1. Find complimentary sequence of reverse primer | 2. Rewrite 5' to 3' of complimentary sequence

Question 57

EB buffer (elution buffer)

Answer 57

Elution efficiency is strongly dependent on the sale concentration and pH of the elution buffer

Question 58

QG buffer (dissolve agarose and adjust pH)

Answer 58

Solubilizes the agarose gel slice and provides the appropriate conditions for binding of DNA to the silica membrane

Question 59

PE buffer (wash filter)

Answer 59

Washes away salts

Question 60

SOC medium

Answer 60

Super optimal broth is used as a recovery medium during the final stage of transformation of E. coli

Question 61

Ampicillin

Answer 61

Prevents growth of non-transformed bacteria

Question 62

IPTG

Answer 62

Compound is a molecular mimic of allolactose Used to induce protein expression where the gene is under the control of the lac operator

Question 63

X-Gal

Answer 63

To test for the presence of an enzyme

Question 64

Alkaline protease

Answer 64

Used to inactivate endonucleases

Question 65

Plant results

Answer 65

Cucumber, Tobacco, green bean, quinoa