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Molecular Biology and Genetics > Restriction mapping > Flashcards

Restriction mapping Flashcards

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1
Q

How can we construct maps?

A

From linear fragments of DNA.

From circular DNA.

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2
Q

What is it useful in genetics?

A

To cut DNA in restrictions.

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3
Q

What is restriction mapping like?

A

Solving a puzzle.

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4
Q

How do we put restriction mapping in action?

A

Cut 2 enzymes –> run them on agarose gel with smaller fragments –> take pic of the information on the gel.

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5
Q

What does each enzyme give us on the agarose gel?

A

Fragments.

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6
Q

What do we do with the fragments and the results we see on the agarose gel?

A

Compare and test the restriction sides.

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7
Q

Why can we not solve maps sometimes?

A

Because they do not give enough information on the agarose gel.

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8
Q

What is it important to have in restriction mapping?

A

A linear map in the end with all the scales of each restriction fragment.

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9
Q

What are the requirements of restriction mapping?

A
  1. Good quality and concentrated DNA.
  2. Accurate size measurements estimations of all the bands that were digested in the gel.
  3. All the combinations of un-digested, single and multiple-digestions measured.
  4. Use of additional Restriction enzymes.
  5. Know if the map is linear DNA fragment or circular.
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10
Q

Why do we need good quality and concentrated DNA for the restriction mapping?

A

To produce a good gel.

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11
Q

Why do we need to have accurate size measurements estimations in restriction mapping?

A

To add up all the digests of all the bands to the same total length of DNA.

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12
Q

Why do we need to use additional restriction enzymes in restriction mapping?

A

To resolve some sections of the map where there is insufficient information to orientate/place fragments.

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13
Q

Why do we need to do sometimes if we do not have the correct information of fragments in restriction mapping?

A

Go back and get the information for the bands.

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14
Q

How many fragments does 1 restriction enzyme give us?

A

1.

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15
Q

How many fragments do 2 restriction enzymes give us?

A

2.

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16
Q

Which are the 3 steps to restriction mapping?

A
  1. DNA Gel & Band Sizing.
  2. Fragments.
  3. Possible order & maps.
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17
Q

What should we do in the first step of restriction mapping?

A

Do the restriction enzymes’ digestion.

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18
Q

What should we do in the Fragment step of the restriction mapping?

A

Run the gel.

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19
Q

What should we do in the ‘Possible order & maps’ step of restriction mapping?

A

Take measurements = results.

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20
Q

What are the DNA fragments?

A

DNA ladders.

21
Q

What should we know exactly about the DNA ladders?

A

Their size.

22
Q

What are the fragments most of the time?

A

As accurate as we need them to be.

23
Q

What should we make sure when we take our results form the restriction mapping process?

A

All the bands are adding up to the same final length. (1000bp for example).

24
Q

How are some RE maps characterised?

A

Simple.

25
Q

How can some RE maps be done?

A

As mental images.

26
Q

What is it easier to do instead of mental images of RE?

A

Draw various versions of the map –> building complexity –> discarding the clearly wrong maps.

27
Q

How can we help our selves with RE maps?

A

Cut blocks of paper –> represent fragments –> move them until the map is solved.

28
Q

What papers can we cut for RE maps?

A

A circular map for RE digest.

29
Q

What can we find from the circular maps we create which have each RE digest?

A

How many times each RE cuts the map.

How many kilobases is each RE.

30
Q

How should we draw our RE in the circular maps?

A

In scale.

31
Q

What should we do after we cut our circular maps for each RE digest?

A

Add the circles together.

32
Q

Where should single RE maps be when adding the circles together?

A

On the inside.

33
Q

Where should the double RE map be when adding the circles together?

A

On the outside.

34
Q

How should we align one RE in the circles when we add them together?

A

Across all three circles.

35
Q

What should all circles have?

A

A cut at 12 o clock.

36
Q

What should we do in order to achieve having all of the RE sites align across all three circles?

A

Rotate the inner circle until all of them are aligned.

37
Q

For what do we draw a circular restriction map?

A

For the sets of fragments from the RE we are putting on an agarose gel.

38
Q

What should all of the bands be?

A

Labelled.

39
Q

What happens in the end of the restriction mapping?

A

All the bands are digested together.

40
Q

How can we solve the restriction mapping puzzle?

A
  1. Find if a linear/circular map is expected.
  2. Find the size of the uncut DNA molecule.
  3. Draw circles with sizes marked in.
41
Q

How can we solve the restriction mapping puzzle?

A
  1. Find if a linear/circular map is expected.
  2. Find the size of the uncut DNA molecule.
  3. Draw circles with sizes marked in.
  4. Make initial maps for single REs.
  5. Overlap the 2 maps.
  6. Rotate the maps until it explains the fragments produced by the 2 REs when digested.
  7. Make a map for the last RE.
  8. Overlap the last RE map on the map of the other 2 REs we already did in step 5.
  9. Choose the correct map combination.
  10. Check if additional information is needed.
42
Q

From where can we find the size of the uncut DNA molecule?

A

From the lanes with the fewest/single band.

43
Q

What kind of DNA pieces can we cut?

A

Singular or circular.

44
Q

How many fragments does a linear restriction cut?

A

2.

45
Q

How many fragments does a circular restriction cut?

A

1.

46
Q

What do we need to get correct in restriction mapping experiment to go well and not have to start again?

A

Incubation: correct pH, enough enzyme.

47
Q

What do we mean by ‘Restriction Mapping’?

A

Have a plasmid –> Digest it with restriction enzymes –> interpret the bands –> draw a map of the plasmid from those bands.

48
Q

What is Lade 1 in the gel of our samples in the restriction mapping?

A

The DNA ladder.

49
Q

What is Lane 2?

A

Undigested ladder.

Molecular Biology and Genetics (15 decks)

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