Mol Methods Lab Review

Question 1

This gene encodes a protein called the PTC receptor, which is found on the surface of taste receptors on the tongue

Answer 1

TASR38 gene

Question 2

Sequence of events for Identifying gene mutations which cause a behavioural abnormality in the fruitfly Drosophila melanogaster

Answer 2

Design primers to amplify the relevant sequence of the target gene -> Perform a PCR using Drosophila genomic DNA as template -> Check the success of the reaction by agarose gel electrophoresis -> Purify & measure the yield of the PCR product -> Ligate the PCR product into a prepared plasmid vector -> Transform bacteria with the vector containing the PCR product -> Grow the transformed bacteria in liquid culture then harvest & purify the plasmid -> Check the identity of the insert by restriction digest & agarose gel electrophoresis -> Use computer based analysis to compare the sequence of the insert with other database entries

Question 3

Fruitfly scientific name

Answer 3

Drosophila melanogaster

Question 4

Sequence of events for Detection & Quantification of Zika virus

Answer 4

Design primers to amplify a relevant Zika gene sequence -> Perform real time qPCR alongside conventional PCR, using cDNA from patient samples -> Check success of conventional PCR by agarose gel electrophoresis -> Analyse real time PCR output data to determine viral load

Question 5

The reaction conditions of a PCR amplification are composed of… and …

Answer 5

total number of cycles to be run & the temp & duration of each step in those cycles

Question 6

PCR conditions

Question 7

PCR conditions

Question 8

What is the connection between the extension time and the size of the PCR product?

Question 9

Is an extension time of 1 minute at 72°C sufficient to amplify a PCR product of 750 base pairs?

Question 10

At what cycle number does the desired double-stranded PCR product appear?

Question 11

What will happen during the PCR if the annealing temperature is too low?

Question 12

What happens if the extension temperature is too high?

Question 13

Components of a PCR

Question 14

What is a PCR ‘master mix’ & what is the advantage of using it?

Question 15

What is the significance of using Taq DNA polymerase in s PCR?

Question 16

What is the function of Magnesium Chloride (MgCl2) in the reaction?

Question 17

The template used in your PCR reaction can either be genomic DNA (gDNA) or complementary DNA (cDNA). What is the difference between these?

Question 18

Thin-walled eppendorfs are more costly to use than conventional tubes. Why do you think they are important for PCR?

Question 19

Use the sequence & primer information of the gene to calculate the expected size of the PCR fragment

Answer 19

750 bp

Question 20

What is the purpose of the DNA cleanup stage of the process?

Question 21

Was your PCR successful?

Question 22

Given the relative sizes of pBluescript (2.96 kb) and the PCR product (0.75 kb) what insert:vector molar ratio have we set up in tube 1?

Question 23

What is the advantage of having different restriction sites at either end of our PCR fragment?

Question 24

What does DNA ligase do in the ligation reaction?

Question 25

If you had a purified stock of DNA & you dilute 1 μL of this purified DNA sample into a total volume of 50 μL distilled water.

Question 26

What other methods of cloning are you aware of

Question 27

What is meant by “competent” cells?

Question 28

Why are the plates incubated upside down?

Question 29

Why is the expression step important?

Question 30

Cells were plated onto a plate without ampicillin. What do you expect to see on this plate tomorrow and why is this control important?

Question 31

Which of the control plate(s) tell you that the antibiotic selection worked? Did this selection work on your own plate(s) and for the majority of the class?

Question 32

Picograms to Nanograms to Micrograms

Question 33

Suppose you have a stock concentration of ampicillin at 100 mg/ml and you want to make 5 ml of L-broth having 50 μg/ml. What volume of stock ampicillin should you add?

Question 34

How much of a 20 mg/ml stock solution of ampicillin would you need to add to 1 litre of L-broth to achieve a final concentration of 100 μg/ml ampicillin?