Questions

Question 1

What defines a colour morph

Answer 1

Variations of the same species which differ by the content of GFB like proteins which determine the different colour of fluorescence.

Question 2

How to study genetic differentiation of the different coour morphs

Answer 2

By coalescent analyses, using 5 markers designed for genes whose expression is modified under stress.

Question 3

Key steps in experimental strategy used to study genetic differentiation across morphs

Answer 3

Sample, extraction, PCR and sequencing, analysis, tree construction

Question 4

Genetic analysis, experimental strategy for DNA extraction

Answer 4

Remove sample from organism and dry, to reaction tube.
Add DNA extraction buffer containing SDS
Pestle, keep in the liquid, don’t draw in air
Add phenol and shake vigorously.
Transfer aqueous phase to a new reaction tube with chloroform, mix, centrifuge for 5
Transfer supernatant to new tube with isopropanol, mix and centrifuge at 1000rpm for 5
Remove supernatant, don’t disturb pellet
Add EtOH (70%) to wash the pellet, centrifuge at 1000rpm for 5.
Centrifuge at max for 20 mins
Carefully remove supernatant and invert the tube on absorbent paper to dry the pellet.
Dissolve pellet in TAE

Question 5

Electrophoresis: no DNA visible

Answer 5

Agarose gel melted.
Buffer has been omitted, or high voltage has been run for too long.
TBE should be used not TAE.
No ethidium bromide added.

Question 6

Electrophoresis, bands too close

Answer 6

Agarose conc is too low for the fragment size
Higher agarose concentration (2-3%) is better for separating smaller fragments
Lower agarose concentration (0.5-1%) is better for separating larger fragments

Question 7

Electrophoresis, poor resolution

Answer 7

Improper choice of sample DNA conc.
Low % gels for high molecular weight DNA and vice versa.
Occurs due to diffusion of DNA through gel particularly at low V for long times.

Question 8

Electrophoresis, band smearing

Answer 8

Overloading the DNA sample or running gels at high V.
Samples loaded into torn wells also causes this, as DNA will tend to run in the interface between the agarose and the gel support

Question 9

Universal primers

Answer 9

primers that can be used to amplify a particular DNA fragment candidate across different samples.
Not 100% complementary to each candidate but enough to prime DNA synthesis.