Restriction mapping Flashcards
How can we construct maps?
From linear fragments of DNA.
From circular DNA.
What is it useful in genetics?
To cut DNA in restrictions.
What is restriction mapping like?
Solving a puzzle.
How do we put restriction mapping in action?
Cut 2 enzymes –> run them on agarose gel with smaller fragments –> take pic of the information on the gel.
What does each enzyme give us on the agarose gel?
Fragments.
What do we do with the fragments and the results we see on the agarose gel?
Compare and test the restriction sides.
Why can we not solve maps sometimes?
Because they do not give enough information on the agarose gel.
What is it important to have in restriction mapping?
A linear map in the end with all the scales of each restriction fragment.
What are the requirements of restriction mapping?
- Good quality and concentrated DNA.
- Accurate size measurements estimations of all the bands that were digested in the gel.
- All the combinations of un-digested, single and multiple-digestions measured.
- Use of additional Restriction enzymes.
- Know if the map is linear DNA fragment or circular.
Why do we need good quality and concentrated DNA for the restriction mapping?
To produce a good gel.
Why do we need to have accurate size measurements estimations in restriction mapping?
To add up all the digests of all the bands to the same total length of DNA.
Why do we need to use additional restriction enzymes in restriction mapping?
To resolve some sections of the map where there is insufficient information to orientate/place fragments.
Why do we need to do sometimes if we do not have the correct information of fragments in restriction mapping?
Go back and get the information for the bands.
How many fragments does 1 restriction enzyme give us?
1.
How many fragments do 2 restriction enzymes give us?
2.
Which are the 3 steps to restriction mapping?
- DNA Gel & Band Sizing.
- Fragments.
- Possible order & maps.
What should we do in the first step of restriction mapping?
Do the restriction enzymes’ digestion.
What should we do in the Fragment step of the restriction mapping?
Run the gel.
What should we do in the ‘Possible order & maps’ step of restriction mapping?
Take measurements = results.
What are the DNA fragments?
DNA ladders.
What should we know exactly about the DNA ladders?
Their size.
What are the fragments most of the time?
As accurate as we need them to be.
What should we make sure when we take our results form the restriction mapping process?
All the bands are adding up to the same final length. (1000bp for example).
How are some RE maps characterised?
Simple.
How can some RE maps be done?
As mental images.
What is it easier to do instead of mental images of RE?
Draw various versions of the map –> building complexity –> discarding the clearly wrong maps.
How can we help our selves with RE maps?
Cut blocks of paper –> represent fragments –> move them until the map is solved.
What papers can we cut for RE maps?
A circular map for RE digest.
What can we find from the circular maps we create which have each RE digest?
How many times each RE cuts the map.
How many kilobases is each RE.
How should we draw our RE in the circular maps?
In scale.
What should we do after we cut our circular maps for each RE digest?
Add the circles together.
Where should single RE maps be when adding the circles together?
On the inside.
Where should the double RE map be when adding the circles together?
On the outside.
How should we align one RE in the circles when we add them together?
Across all three circles.
What should all circles have?
A cut at 12 o clock.
What should we do in order to achieve having all of the RE sites align across all three circles?
Rotate the inner circle until all of them are aligned.
For what do we draw a circular restriction map?
For the sets of fragments from the RE we are putting on an agarose gel.
What should all of the bands be?
Labelled.
What happens in the end of the restriction mapping?
All the bands are digested together.
How can we solve the restriction mapping puzzle?
- Find if a linear/circular map is expected.
- Find the size of the uncut DNA molecule.
- Draw circles with sizes marked in.
How can we solve the restriction mapping puzzle?
- Find if a linear/circular map is expected.
- Find the size of the uncut DNA molecule.
- Draw circles with sizes marked in.
- Make initial maps for single REs.
- Overlap the 2 maps.
- Rotate the maps until it explains the fragments produced by the 2 REs when digested.
- Make a map for the last RE.
- Overlap the last RE map on the map of the other 2 REs we already did in step 5.
- Choose the correct map combination.
- Check if additional information is needed.
From where can we find the size of the uncut DNA molecule?
From the lanes with the fewest/single band.
What kind of DNA pieces can we cut?
Singular or circular.
How many fragments does a linear restriction cut?
2.
How many fragments does a circular restriction cut?
1.
What do we need to get correct in restriction mapping experiment to go well and not have to start again?
Incubation: correct pH, enough enzyme.
What do we mean by ‘Restriction Mapping’?
Have a plasmid –> Digest it with restriction enzymes –> interpret the bands –> draw a map of the plasmid from those bands.
What is Lade 1 in the gel of our samples in the restriction mapping?
The DNA ladder.
What is Lane 2?
Undigested ladder.
