PRACTICAL A: Molecular biology Flashcards
What is the average genome size of a plasmid?
3kb
What is the genome size of E.Coli?
4 Mbp
What is the genome size of a human?
3000 Mbp
How long is the average gene?
~10^4 bp
What is found on DNA between genes?
Large stretches of non-coding DNA
How is the genetic material arranged in animals?
DNA molecules are packaged in a number of chromosomes, found in the nucleus
How is the genetic material arranged in bacteria?
They have a single circular molecule of genomic DNA and a number of “extra-chromosomal” circular molecules, called plasmids, found within the cytoplasm.
When in the bacterial cell, the circular DNA is found as
a) supercoiled molecule
b) relaxed circle
c) liner
a) supercoiled molecule, to allow efficient packaging
What is the state of the circular DNA molecules in the preparations used in the lab?
Some molecules are supercoiled, others become relaxed.
What affects migration patterns on the agarose gel?
The physical properties of the DNA molecules.
This includes both size and condition (supercoiled, more compact, relaxed, etc.)
Supercoiled: more compact, travels faster
What are restriction endonucleases?
Enzymes that cut DNA at specific recognition sequences.
They are a naturally-occurring bacterial defence against viral infection.
Restriction sites are __________ .
Palindromic: read the same backward or forward
How can the frequency with which the recognition sites occur be calculated?
Freq = (1/4)^n
n: number of base pairs in the recognition sequence
How can the lengths of restricted DNA fragments be determined?
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)
What is the relationship between migration distance on gel and size of the DNA molecule?
They are inversely proportional: smaller molecules travel further than bigger molecules.
What is a restriction map?
The restriction map of a DNA fragment can show the positions of the different target sites for various restriction enzymes.
In gel electrophoresis, what are the charges at the cathode and at the anode?
Anode: +ve, red
Cathode: -ve, black
What is the charge on a DNA molecule and where does it come from?
Negative, conferred by phosphodiester backbone at ph >1
How is DNA visualised after running the gel?
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
How is the position of the DNA fragments visualised while running the gel?
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.
What is the composition of the gel buffer used in the experiment?
TBE: Tris base, boric acid, EDTA
Outline the experimental procedure
Preparing mixtures, incubation, preparation of gel, electrophoresis, photograph, analysis of data
More detailed:
- 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)
- Contents of the tube were mixed by vortexing, and then returned to the bottom of the tube by microcentrifuge
- Tubes with DNA and RE were incubated for 30min at 37ºC
- Molten agarose gel was set, gel buffer was added
- Loading dye was added, the digests were added together with the DNA markers
- Gel was electrophoresed at 70V for 40min
- Gel was disconnected from power supply and photograph was taken, plus it was observed under UV light
How can the length of a DNA fragment be determined?
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
What’s on each axis of the graph used to determine the length of the DNA fragments?
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.
