Practical A

Question 1

For this experiment you will be using an agarose gel which is made by melting a specific quantity of agarose in TBE buffer. You will use 50ml of the agarose/TBE solution and this will contain 0.8% agaraose. How much agarose did the technicians measure out for your gel?

Answer 1

0.4g

Question 2

How many base pairs are found in the maize genome?

Answer 2

3 x 10^10 base pairs

Question 3

What is the probability of any four particular bases occurring in a DNA sequence? 1 in…

Answer 3

1 in 256

Question 4

Where are restriction endonucleases naturally found?

Answer 4

Made by micro-organisms

Question 5

Why is glycerol present in the gel loading buffer?

Answer 5

It makes the sample denser than the running buffer so it sinks to the bottom of the well

Question 6

Restriction digests are often set up using a concentrated stock of buffer solution. This would commonly be ten times as concentrated as the working buffer and would be referred to as a 10x buffer. If your digest was to be carried out in a final volume of 40ul how much of your 10x stock buffer would you need to add?

Answer 6

4ul

Question 7

W jakim kierunku gelu spływa DNA

Answer 7

w kierunku positive electrode, bo DNA jest negative

Question 8

Loading dye has 2 essential roles when applying samples to a gel:

Answer 8

the blue color allows you to see where the sample has been added

since solution contains glycerol, which makes it more dense than the running buffer so that it allows your samples to sink easily into the well.

Question 9

What running buffer are we using in this experiment?

Answer 9

TBE

Question 10

What does the running buffer do?

Answer 10

It carries the charge between the electrodes, so that it allows DNA to move in the electric field.

Question 11

Why are we using DNA marker?

Answer 11

So that we can compare the migration of our samples to work out the size of our DNA.

Question 12

What does a DNA marker contain/consist of?

Answer 12

DNA fragments of known sizes

Question 13

Na jak długo i o jakim napięciu trzeba zostawić próbkę, aby zauważyć wyniki?

Answer 13

40 min, 70V

Question 14

Wymień poszczególne kroki w eksperymentu: (13)

Answer 14

1. Stwórz odpowiednie additions (są w wordzie)
2. Mix the contents of the tube by vortexing.
3. Spin in the microcentrifuge to return the contents to the bottom of the tube.
4. Incubate for 30 minutes at 37C.
5. Arrange buffer plates and well comb in appropriate slots.
6. Pour 50ml of molten agarose into the apparatus. Take care and wear gloves, gel contains ethidium bromide.
7. Leave until set.
8. Remove buffer plates and comb.
9. Add TBE (contains ethidium bromide so wear gloves).
10. Add 3l of loading dye to tubes 1-6.
11. Load all (or as much as possible) of your digests and the DNA markers in the pattern shown below. A demonstrator will show you how.

Fill the gel wells in this order:

(a) markers
(b) tube 1: uncut genomic DNA
(c) tube 2: genomic DNA cut with EcoRI
(d) tube 3: genomic DNA cut with AluI
(e) tube 4: uncut plasmid DNA
(f) tube 5: plasmid DNA cut with EcoRI
(g) tube 6: plasmid DNA cut with EcoRI + Bgl II

12. Connect to a power supply and electrophorese at 70V for approx. 40 minutes.
13. Disconnect from the power supply and have a photograph taken of your gel.

Question 15

Why do we have to use gloves in this experiment?

Answer 15

because TBE contains ethidium bromide

Question 16

Alu 1 enzyme:
Isolated from:
Sequence recognised:

Answer 16

1. Arthrobacteria luteus
2. I
   -AG CT-
   -TC GA-
   I

Question 17

Taq 1 enzyme:
Isolated from:
Sequence recognised:

Answer 17

1. Thermus aquaticus YTI
2. I
   -T CGA-
   -AGC T-
   I

Question 18

EcoR 1 enzyme:
Isolated from:
Sequence recognised:

Answer 18

1. Escherichia coli RY13
2.
    I
-G AATTC-
-CTTAA G-
              I

Question 19

Not 1 ENZYME:
Isolated from:
Sequence recognised:

Answer 19

1. Nocardia otitidis-caviarum
2. I
   -GC GGCCGC-
   -CGCCGG CG-
   I