PRACTICAL A: Molecular biology

Question 1

What is the average genome size of a plasmid?

Answer 1

3kb

Question 2

What is the genome size of E.Coli?

Answer 2

4 Mbp

Question 3

What is the genome size of a human?

Answer 3

3000 Mbp

Question 4

How long is the average gene?

Answer 4

~10^4 bp

Question 5

What is found on DNA between genes?

Answer 5

Large stretches of non-coding DNA

Question 6

How is the genetic material arranged in animals?

Answer 6

DNA molecules are packaged in a number of chromosomes, found in the nucleus

Question 7

How is the genetic material arranged in bacteria?

Answer 7

They have a single circular molecule of genomic DNA and a number of “extra-chromosomal” circular molecules, called plasmids, found within the cytoplasm.

Question 8

When in the bacterial cell, the circular DNA is found as

a) supercoiled molecule
b) relaxed circle
c) liner

Answer 8

a) supercoiled molecule, to allow efficient packaging

Question 9

What is the state of the circular DNA molecules in the preparations used in the lab?

Answer 9

Some molecules are supercoiled, others become relaxed.

Question 10

What affects migration patterns on the agarose gel?

Answer 10

The physical properties of the DNA molecules.

This includes both size and condition (supercoiled, more compact, relaxed, etc.)

Supercoiled: more compact, travels faster

Question 11

What are restriction endonucleases?

Answer 11

Enzymes that cut DNA at specific recognition sequences.

They are a naturally-occurring bacterial defence against viral infection.

Question 12

Restriction sites are __________ .

Answer 12

Palindromic: read the same backward or forward

Question 13

How can the frequency with which the recognition sites occur be calculated?

Answer 13

Freq = (1/4)^n

n: number of base pairs in the recognition sequence

Question 14

How can the lengths of restricted DNA fragments be determined?

Answer 14

By means of agarose gel electrophoresis, which separates them according to their size, and subsequent comparison with fragments of known size (DNA markers, also run on the gel)

Question 15

What is the relationship between migration distance on gel and size of the DNA molecule?

Answer 15

They are inversely proportional: smaller molecules travel further than bigger molecules.

Question 16

What is a restriction map?

Answer 16

The restriction map of a DNA fragment can show the positions of the different target sites for various restriction enzymes.

Question 17

In gel electrophoresis, what are the charges at the cathode and at the anode?

Answer 17

Anode: +ve, red

Cathode: -ve, black

Question 18

What is the charge on a DNA molecule and where does it come from?

Answer 18

Negative, conferred by phosphodiester backbone at ph >1

Question 19

How is DNA visualised after running the gel?

Answer 19

With EtBr: it intercalates between bases and takes on a pink/orange fluorescence when illuminated with UV light at 300nm.

The ethidium bromide is present in the TBE buffer

Question 20

How is the position of the DNA fragments visualised while running the gel?

Answer 20

Loading dye. It contains glycerol so that the samples are more dense than the buffer and sink into the well and bromophenol blue which runs ahead of the smallest fragments indicating when the electrophoresis is complete.

Question 21

What is the composition of the gel buffer used in the experiment?

Answer 21

TBE: Tris base, boric acid, EDTA

Question 22

Outline the experimental procedure

Answer 22

Preparing mixtures, incubation, preparation of gel, electrophoresis, photograph, analysis of data

More detailed:

1. Six tubes were prepared: 1-3 with genomic DNA, 4-6 with plasmid DNA. Tubes 1 and 4 were controls (no RE added, just DNA)
2. Contents of the tube were mixed by vortexing, and then returned to the bottom of the tube by microcentrifuge
3. Tubes with DNA and RE were incubated for 30min at 37ºC
4. Molten agarose gel was set, gel buffer was added
5. Loading dye was added, the digests were added together with the DNA markers
6. Gel was electrophoresed at 70V for 40min
7. Gel was disconnected from power supply and photograph was taken, plus it was observed under UV light

Question 23

How can the length of a DNA fragment be determined?

Answer 23

By comparing the electrophoretic migration of a fragment with that of fragments of known length

This can be done

• visually, estimation from the picture
• by drawing a graph

Question 24

What’s on each axis of the graph used to determine the length of the DNA fragments?

Answer 24

X axis: log(bp). Each big square: 0.05bp, starting from 2.6

Y axis: electrophoretic distance migrated (mm). Each big square: 5mm

Assume relationship between these two variables linear, although the plot has quite a lot of scatter and larger fragments may show significant deviations. Draw line of best fit and work from there.