Missed Marks

Question 1

What is the equilibrium reaction for an anion exchanger resin (Resin-OCH2CH2N(CH2CH3)2 ?

Answer 1

Resin-OCH2CH2N(CH2CH3)2 + H+ <-> Resin-OCH2CH2NH+(CH2CH3)2

Question 2

When measuring protein concentration via spectroscopy, why do we use 280 nm specifically?

Answer 2

Trp absorbs specifically at 280 nm; we need a wavelength in the UV spectrum, as the protein is colourless

Question 3

Comment on the reliability of a Km value derived from a Michaelis-Menten plot, and suggest how this could be improved

Answer 3

This is not a very reliable estimate of Km, as the graph has not yet levelled off, so Vmax cannot accurately be determined; use a Lineweaver-Burk plot of Hanes-Wolf plot and determine Km from the intercepts

Question 4

State 2 factors that could affect Km

Answer 4

1. The molecular structure of the enzyme determines how tightly they can bind the substrate
2. Temperature - high temperatures can disrupt the bonds between enzyme and substrate

Question 5

State the controls necessary in a conjugation experiment involving TetR Strain A and NalR Strain B

Answer 5

Plate undiluted strain A onto a Nal (or Nal, Tet) plate - lack of growth will show that there are no cells in the A culture with resistance to Nal

Plate undiluted strain B onto Tet (or Nal, Tet) plate - lack of growth will show that there are no cells in the B culture with resistance to Tet

Question 6

In an experiment on horizontal gene transfer of Resistance genes, which genes are most likely to be plasmid encoded - the ones that are transferred or the ones that are not?

Answer 6

The ones that are transferred - plasmid transfer is the most likely option for horizontal gene transfer

Question 7

When looking at SDS-PAGE results, why are some bands thicker than others

Answer 7

The thickness of the band is due to the amount of protein binding the dye - more protein results in thicker, darker bands

Question 8

How would you tell which gel has the lowest polyacrylamide % ?

Answer 8

If the bands have migrated further - especially if certain light chain bands have migrated OUT of the gel into the buffer

Question 9

What are gel 3 (lower PA %) and gel 4 respectively useful for?

Answer 9

Gel 3 is useful for resolving and comparing LARGE proteins, while 4 is useful for SMALLER proteins

Question 10

State how you can tell a particular PCR result is a VNTR test?

Answer 10

A range of molecular weights of bands, because the number of repeats varies per person - used in forensics because of the huge amount of possible alleles

Question 11

State how you can tell a particular PCR result is a TE test?

Answer 11

There are only 2 possible bands - patients either have the insert (heavier band) or they don’t

Question 12

State how you can tell a particular PCR result is an SNP test?

Answer 12

Restriction enzyme digestion before loading - this shows the method was PCR followed by RFLP, which is typical of testing for SNPs

Question 13

State why the gel in the Cub Paternity Test post-lab might have led to ambiguous results, and 2 ways to improve this

Answer 13

Some bands were too close together, making them hard to distinguish (adapt the gel to better discriminate using tighter pores; OR redesign primers so PCR fragment includes HpaI recognition site, then no need for BamHI, and fragments will be larger so less ambiguous

Question 14

2 things missing from WT vs delta-Rad9 graph

Answer 14

TITLE and use legend to distinguish two groups (e.g., dots and triangles)

Question 15

What is the key phrase to use when stating that Rad9 has been mutated?

Answer 15

NO FUNCTIONAL COPY OF RAD9

Question 16

In wild type S. cerevisiae, what happens when DNA is damaged?

Answer 16

The cell cycle is halted

Question 17

What actually causes the lower % survival in mutants when exposed to radiation?

Answer 17

The increase in mutations leads to cells being unsustainable and cell death, hence lower % survival

Question 18

How would you transform the raw survival data and present it graphically?

Answer 18

Convert raw survival numbers into % survival, calculate average % survival (from 8 groups) as well as the SD/SEM, then plot as a bar graph with error bars (UV-dose, x, vs % survival, y)

Question 19

Which statistical analysis would be appropriate to compare mean survival % at 0, 20, 40, 60, 80 J m-2?

Answer 19

Kruskal-Wallis test -> comparing means of more than two groups, but the data is NON-PARAMETRIC because it is a percentage

(Also post hoc Dunn’s apparently?)

Question 20

Which statistical analysis would be appropriate to compare mean survival % between WT and non-WT?

Answer 20

Mann-Whitney U test - comparing means of two groups, no paired data, non-parametric because data are ordinal

Question 21

Why is the INITIAL rate of reaction measured in enzyme kinetics experiments?

Answer 21

Assuming E + S form a complex in the reaction which breaks down to give P, at the start of the reaction no product will have been formed yet, so the rate of the reverse reaction is negligible

THEREFORE, we know the exact concentration of substrate that gives the observed rate

Question 22

What are the major steps of a PCR/RFLP experiment?

Answer 22

1. Extract DNA (e.g., from cheek cells)
2. PCR
3. Digest with Restriction Enzymes
4. Agarose Gel Electrophoresis to reveal Restriction Fragment Length Polymorphisms