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Biotechnology I Lab > Exam 2 > Flashcards

Exam 2 Flashcards

1
Q

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

A

Grinding

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2
Q

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

A

Lysis

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3
Q

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

A

Temperature, pH, and time

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4
Q

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

A

Precipitant

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5
Q

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

A

Binds to the column

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6
Q

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

A

Analyzing

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7
Q

The purpose for washing a column containing bound DNA is?

A

Washing away impurities

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8
Q

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

A

Eluting

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9
Q

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

A

GoTaq, forward primer, reverse primer

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10
Q

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

A

Competent

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11
Q

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

A

Forward and reverse primers

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12
Q

Primers consist of ___.

A

Oligonucleotides

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13
Q

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

A

Taq polymerase

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14
Q

There are three primary temperature changes in PCR reaction:

A

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

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15
Q

The temperature a PCR product should be stored at is?

A

4C

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16
Q

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

A

Thermocycler

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17
Q

What does PCR stand for?

A

Polymerase chain reaction

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18
Q

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

A

Gel electrophoresis

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19
Q

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

A

Agarose gel

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20
Q

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

A

Excision

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21
Q

A “ladder” is also known as..

A

Gene ruler or molecular ruler

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22
Q

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

A

Band

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23
Q

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

A

Charge and pH

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24
Q

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

A

7.5

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25
Q

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

A

Lower

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26
Q

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

A

pGEM-T

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27
Q

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

A

Overhangs

28
Q

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

A

LacZ

29
Q

What does the LacZ gene interact with?

A

B-galactosidase

30
Q

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

A

Transformation

31
Q

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

A

P-shock

32
Q

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

A

42C

33
Q

After heat shocking, how was the reaction quenched?

A

Ice bath

34
Q

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

A

E. coli

35
Q

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

A
  • 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
36
Q

What is T4 DNA ligase used for?

A
  • catalyzes the formation of a phosphodiester bond
  • repairs single-strand nicks
  • joins DNA fragments with either cohesive or blunt termini
37
Q

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

A

Shearing of the DNA

38
Q

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

A

SOC medium

39
Q

What temperature were the transformed cells incubated at?

A

65C

40
Q

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

A

E. coli, part of plasmid, rubisco

41
Q

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

A

Sp6 and T7

42
Q

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

A

599-750

43
Q

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

A

150

44
Q

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

A
  • 150 would be plasmid

- 750 would be gene+plasmid

45
Q

rbcL

A

Codes for large subunits of rubisco

46
Q

GoTaq green master mix

A 
Taq polymerase
MgCl2
dNTPs
Loading buffer
Green loading dye
47
Q

MgCl2

A

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

48
Q

dNTPs

A

Free nucleotides to add to growing fragment

49
Q

Loading buffer

A

For better amplification of GC and AT rich targets

50
Q

Green loading dye

A

Allows tracking during gel electrophoresis

51
Q

Buffer AW1

A

Binding buffer

52
Q

Buffer AW2

A

Wash buffer

53
Q

Buffer AE

A

Elution buffer

54
Q

Buffer AP1

A

Lysis buffer

55
Q

Buffer P3

A

Neutralization buffer for plasmid preparation

56
Q

PCR with primers

Two steps

A
  1. Find complimentary sequence of reverse primer

2. Rewrite 5’ to 3’ of complimentary sequence

57
Q

EB buffer (elution buffer)

A

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

58
Q

QG buffer (dissolve agarose and adjust pH)

A

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

59
Q

PE buffer (wash filter)

A

Washes away salts

60
Q

SOC medium

A

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

61
Q

Ampicillin

A

Prevents growth of non-transformed bacteria

62
Q

IPTG

A

Compound is a molecular mimic of allolactose

Used to induce protein expression where the gene is under the control of the lac operator

63
Q

X-Gal

A

To test for the presence of an enzyme

64
Q

Alkaline protease

A

Used to inactivate endonucleases

65
Q

Plant results

A

Cucumber, Tobacco, green bean, quinoa

Biotechnology I Lab (3 decks)

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