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Molecular Biology Lab. > Lab 4. > Flashcards

Lab 4. Flashcards

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1
Q

What are restriction enzymes?

A

Enzymes that will recognise and cut a short palindromic DNA sequence.

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2
Q

Do restriction enzymes cut one DNA strand or both DNA strands?

A

They cut both DNA strands.

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3
Q

What is the specific DNA sequence that is cleaved by a restriction enzyme known as?

A

The recognition site.

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4
Q

Where is the cleavage site for a restriction enzyme located?

A

It is usually located in or near to the recognition sequence.

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5
Q

The restriction enzyme ECO-R1 cleaves at what DNA sequence?

A

GAATTC.

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6
Q

BAM H1 is derived from which organism?

A

Bacillus amyloliquefaciens.

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7
Q

ECO-R1 is derived from what organism?

A

Escherichia coli RY 13.

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8
Q

HIND-III is isolated from which organism?

A

Haemophilus inflenzae Rd .

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9
Q

MBO-1 is isolated from which organism?

A

Moraxella bovis.

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10
Q

PST-1 is isolated from which organism?

A

Providencia stuartii.

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11
Q

SMA-1 is isolated from which organism?

A

Serratia marcescens.

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12
Q

TAQ-1 is isolated from which organism?

A

Thermophilus aquaticus.

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13
Q

How are restriction enzymes named?

A

The 1st 3 letters are the first 3 letters from the organism that they were discovered in.

The number refers to the order that they were found in.

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14
Q

How are sticky ends created?

A

By leaving overlapping fragments of DNA on each strand so that the 2 strands can re-join easily.

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15
Q

Why is the location of the cleavage sites of restriction endonucleases important?

A

Because this kowledge helps with analysis and mapping of DNA segments in molecular cloning experiments.

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16
Q

What does restriction mapping involve?

A

It involves working out the positions of the cleavage sites in relation to one another within a DNA molecule.

17
Q

How is restriction mapping done?

A

By determining the size of the DNA fragments generated by different combinations of restriction enzymes.

18
Q

What is the first step of constructing a restriction map?

A

To determine the number of fragments each individual enzyme produces.

19
Q

How is the size and number of frgaments from a DNA molecule determined?

A

By agarose gel electrophoresis.

20
Q

How are the distances between cleavage sites determined?

A

Via reactions using various combinations of enzymes and then determining the distances between cleavage sites.

21
Q

How are the relative positions of the cleavage sites ordered?

A

In a way that is consistent with all of the observed fragment sizes.

22
Q

Gel electrophoresis can determine the size of DNA fragments that are between what size?

A

Between 500 and 30,000 base pairs.

23
Q

How does the agarose gel separate DNA fragments into an order of size?

A

The gel contains microscopic pores that act as
a molecular sieve.

The smaller DNA molecules will move quickly through the gel whereas the large molecules will move slowly.

This means that the small fragments will move further through the gel than the large fragments.

24
Q

What is combined with the gel in the electrophoresis chamber?

A

A buffer solution and electrodes.

25
Q

What part of the electrophoresis chamber will DNA migrate to?

A

It will migrate towards the positive electrode as it has a negative charge.

26
Q

How does electrophoesis separate the DNA fragments?

A

By their size and shape.

27
Q

What part of the DNA molecule gives DNA its negative charge?

A

The negatively charged phosphodiester bonds.

28
Q

How many phosphate groups are their per base pair in a DNA molecule?

A

For every base pair there are 2 charged phosphate groups.

29
Q

How can DNA fragments of an unknown size be determined after electrophoresis?

A

They are electophoresed next to Standard DNA fragments of known size which act as markers.

30
Q

What is measured once migration has finished?

A

The migration distances of the known and unknown fragments are measured.

31
Q

How is duplex DNA detected?

A

By staining the DNA with intercalating substances between the stacked base pairs.

32
Q

What are 3 common intercalating substances?

A

Ethidium.

Acridine orange.

Proflavin.

33
Q

Which stain exhibit flurorescence under UV light?

A

SYBR green.

34
Q

What reagents were added to the testube in lab 4?

A

The reaction cocktail.

Water.

ECO-R1.

BAM-H1.

Molecular Biology Lab. (7 decks)

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