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methods > Midterm 5 > Flashcards

Midterm 5 Flashcards

1
Q

What kind of vector is used in subcloning? Why?

A

An expression vector to establish the goal of expressing the gene

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2
Q

What insert is used from sublconing?

A

The same vector obtained from cloning.

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3
Q

What type of expression is done in subcloning?

A

Overexpression

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4
Q

Define subcloning

A

Transferring a insert from a cloning vector to an expression vector and to overexpress the gene of itnerest

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5
Q

Why can only DNA polymerase be used in cloning?

A

The cloning vector does not have a promoter

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6
Q

What type of polymerase can be used in a subcloning vector? Why?

A

RNA pplymerase because the expression vector has a promoter.

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7
Q

What can be obtained from a expression vector?

A

mRNA to be translated into protein

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8
Q

How does RNA polymerase work in the expression vector?

A

It binds to the promoter of the vector for transcription.

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9
Q

What are the features of a cloning vector?

A

MCS/ polylinker/restriction site, ORI, and selectable marker

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10
Q

What are the features of a expression vector?

A

MCS/ polylinker/restriction site, ORI, and selectable marker, promoter

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11
Q

What are we transferring in subcloning in the present experiment? Also name the vectors?

A

Transferring CR insert from PGEMT Easy Vector to Pet29b Vector

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12
Q

What is one way to transfer the insert to the expression vector.

A

Use restriction enzymes to “cut” the insert from the cloning vector and ligated to the expression vector

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13
Q

What do you need to make sure about the restriction sites of the CR insert?

A

he insert does not have the same restriction sites within the gene as the restriction enzyme you are using, only at the ends

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14
Q

What do the restriction enzymes use have to match on the vector?

A

Restriction sites

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15
Q

What type of restriction endonucleases do we use?

A

2

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16
Q

What type of ends do the restriction enzymes need to give?

A

Sticky ends

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17
Q

How are restriction sites added to an insert if they are not present?

A

Add restriction sites by adding sequences of DNA that are cut by the restriction sites on the expression site through PCR primers. Forward primer corresponds to one restriction site and reverse to another.

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18
Q

What do the forward and reverse primers in subcloning PCR contain.

A

Forward primer contains a sequence of nucleotides matching the restriction site of a restriction enzyme flanking one end of the vector and the reverse contains another sequence of nucleotides matching the restriction site of a restriction enzyme flanking the other end of the vector.

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19
Q

What will happen during annealing in the subcloning PCR.?

A

The primers will anneal and fragments will containing the restriction sites corresponding to the restriction enzymes in the vector.

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20
Q

True or False: The primers are the same as used in cloning PCR.

A

False. Parts of them are the same but the restriction sites are added.

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21
Q

Why can primers vary in subcloning?

A

for adjusting the melting temperature.

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22
Q

What approach/ strategy is used to prepare the vector and insert for ligation

A

Restriction digestion through double digestion

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23
Q

How are restriction sites added to the insert? Where are they added?

A

PCR. 5’ and 3’ of the inser

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24
Q

What restriction sites are added to the insert in the present subcloning PCR experiment?

A

NdeI and XhoI

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25
Q

Why were the two restiction enzymes chosen in this experiment and how?

A

We performed an insilico analysis of restriction sites or the CR gene to determine the restriction enzymes to use for double digestion, to cut the CR insert. We chose NdeI and XhoI because the restriction sites corresponded to those present in our pet29b vector and not within the CR gene, so the insert would not be cut. Furthermore, we wanted to identify restriction sites that kept out insert in the 5’ 3’ orientation so at the 5’ 3’ of the MCS on the vector, and that cut sticky ends.

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26
Q

Why is what we used to cut the insert and vecetor called double digestion?

A

Because two enzymes are used.

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27
Q

How is the expression vector supplied?

A

As a circular vector

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28
Q

What dou you need to do to the expression vector and the insert so ligation can happen? Name the experiment.

A

Cut them with the same restriction enzymes through double digestion.

29
Q

Why are we using two enzymes?

A

to prevent the vector from self ligating (non complementary sticky ends).
Insert orientation can also be incorrect since the restriction sites are the same and can be inserted, flipped. Can result in the promoter being at the wrong end, preventing directional cloning

30
Q

What type of cloning are we trying to do with the insert in the subcloning vector? Name and define it.

A

directional cloning: cloning to maintain the ORF of the insert.

31
Q

What is done in ligation in subcloning?

A

Joining of the vector and the insert through the use of DNA ligase facilitating the formation of a phosphodiester bond between the vector and ends of the insert.

32
Q

What type of proofreading activity is done by polymerase in subcloning? What is the goal?

A

Exonuclease activity: modifies at the ends of the DNA fragment.
Goal is to remove mutations

33
Q

What is the vector used in cloning, subcloning?

A

PGEMT-Easy vector; Pet29bCR (expression vector)

34
Q

how is the vector supplied in cloning, subcloning?

A

linear vector with T overhangs, circular vector with restriction sites to cut into a linear vector

35
Q

What experiment is done to prepare the vector in cloning, subcloning?

A

None. Double digestion to cut the vector linear with sticky ends.

36
Q

Describe the insert in cloning, subcloning.

A

Insert (CR ORF) has A overhangs at the 3’, done by taq polymerase; CR ORF needs to have restriction sites added at the 5’ and 3’. Need to create the sticky ends with the same restriction sites as the restriction enzyme used to cut the vector (NdeI and XhoI)

37
Q

what is the goal in cloning, subcloning?

A

Goal is to make numerous identical copies; Goal is overexpression of the gene of interest

38
Q

What type of polymerase do we use in cloning, subcloning? Do they have proofreading activity?

A

Use taq DNA polymerase. Taq polymerase does not have proofreading activities; pfu Turbo polymerase with proofreading activities

39
Q

Why do we not want proofreading activity in cloning?

A

Do not want proofreading activity due to the activity being exonuclease and will remove overhangs.

40
Q

Why do we want proofreading activity in subcloning?

A

Due to the goal of your PCR mainly being to attach restriction sites to your insert, the insert does not need to have overhangs since we will be using double digestion to cut the insert to create overhangs.
However the polymerase we use must have proofreading activity. The insert will be expressed into a protein. Therefore if there is a mutation, it will affect protein expression.

41
Q

Does the cloning and or subcloning vector have blue/white screening capability? Why or why not?

A

Cloining does due to presence of Lac Z’ gene, broken when the insert is ligated.
The subcloning vector does not due to not having the LacZ’ gene.

42
Q

Does cloning and or subcloning vector have control elements in the vector. Which ones do they have.

A

Cloning does not. The subcloning vector has: promoter, operator, rbs, thrombins site, s tag, his tag

43
Q

What antibiotic resistance is in the cloning, subcloning vector?

A

Ampicillin; Kanamycin.

44
Q

Does the cloning; subcloning vector need to be purified? How and why?

A

The cloning vector does not need to. The subcloning vector needs to through gel purification due to double digestion, done to cut the vector.

45
Q

Draw the pet29b vector

A
46
Q

What is the product of a transcription vector; translation vector?

A

Gives a transcript (mRNA)
; Gives a sequence of amino acids.

47
Q

How is the vector determined to be a transcription vector?

A

A suffix following the name.

48
Q

How is a vector chosen? What can happen if it is not chosen correctly?

A

by looking at insert. Want to make sure the insert and the vector match. If it does not match, a frameshift mutation can occur and affect protein expression.
Ex: The CR gene starts with ATG so my vector needs to start at TAC to bind.

49
Q

What are the control elements on the expression vector?

A

Promoter, Rbs, S-tag, thrombin/cleavage site, his tag

50
Q

What is the T7 promoter? Why is needed? Where does it come from? What happens if it is removed

A

Promoter is necessary for binding of RNA polymerase for transcription.
Comes from bacteriophage T7
T7 RNA polymerase binds to it
If removed, no transcription.

51
Q

What is the RBS? What is it needed for? What type of element is it? What does it allow for?

A

Ribosome binding site
Necessary for translation
Translation initiation element
allows for efficient production of the protein of interest

52
Q

What is an S tag? What Does it allow for? How many AA is it and what type of charge? What does it improve? When do you want to use it? Do we need it in the present experiment, why or why not?

A

An s tag is a tag so it can be tracked. Some times we obtain a lot of proteins so it can used to track the protein of interest. It also helps the detection of non-pure elements in purification by using a column made for s-tag purification. It is 15AA of charged polar amino acids. It improves the solubility of the protein and used when you want a soluble protein. We remove it during restriction digestion because CR is soluble

53
Q

How large is the thrombin site in AA and what does it do?

A

Made up of 6 AA
Cleaves by breaking the peptide bond between two amino acids, arginine and glycine.

54
Q

What is the bond between two amino acids?

A

Peptide bond

55
Q

What does the MCS contain?

A

Contains uniquely present restriction sites

56
Q

What is the Lac Operator? What is called on the vector? When is it necessary? What is it based on? What does it do?

A

Lac I
Necessary when using E.coli host cells
Based of lac operon
Acts like a switch initiating the transcription.

57
Q

How many histidine are on the His Tag? What does it help with? What does it act like and why is this useful?

A

6 histidine
Helpful for purification
Acts like a detector for detecting the protein of interest. Helps verify protein is successfully expressed

58
Q

What is the order of the control elements in a vector before it is cut?

A

Promoter: T7 Promoter
Operator: Lac Operon
XbaI
RBS: Ribosome Binding Site
Nde1
sTAG/ TAG site
Thrombin Site
MCS
XhoI
His tag
Stop Codon

59
Q

What is the order of the control elements in a vector after it is cut?

A

Promoter: T7 Promoter
Operator: Lac Operon
XbaI
RBS: Ribosome Binding Site
insert
His tag
Stop Codon

60
Q

What type of ends can restriction enzymes cut? How do you know?

A

Restriction enzymes can cut sticky or blunt ends
Know from how the errors are pointed
If the arrows are aligned, blunt ends
If the arrows are not aligned, sticky ends

61
Q

When do protruding ends occur? What is a trick for determining the protruding end?
Practice protruding ends

A

Trick for protruding ends is to look at the end with less nucleotides and where it starts.
Protruding ends only occur for sticky ends.

62
Q

How many bp should a primer be? Why do you want this size?

A

Want a primer 18-20bp
- Want this size because if the primer is too small it can bind to other regions within the DNA and not at the ends
- If the primer is too long, there is a higher chance it can bind to itself and the melting temperature can increase.

63
Q

How do you determine the primer sequence?

A

Do a blast

64
Q

What percentage of GC do you want? Why?

A

40-60%
GC makes it more stable

65
Q

What nucloeotides do you want to start your primer with, what is it called and why do you want it?

A

Start with GC, called the GC clamp to make it more stable at the beginning and the end

66
Q

What is the tm difference you want between the RP and FP?

A

5 degrees.

67
Q

What does the annealing temeperature depend on

A

the melting temperature of the primers and the length of the primer.

68
Q

True or False: Primer should not have complementary regions within the primer or between the FP and RP

A

True

methods (14 decks)

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