Mol Methods Lab Review Flashcards
This gene encodes a protein called the PTC receptor, which is found on the surface of taste receptors on the tongue
TASR38 gene
Sequence of events for Identifying gene mutations which cause a behavioural abnormality in the fruitfly Drosophila melanogaster
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
Fruitfly scientific name
Drosophila melanogaster
Sequence of events for Detection & Quantification of Zika virus
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
The reaction conditions of a PCR amplification are composed of… and …
total number of cycles to be run & the temp & duration of each step in those cycles
PCR conditions
PCR conditions
What is the connection between the extension time and the size of the PCR product?
Is an extension time of 1 minute at 72°C sufficient to amplify a PCR product of 750 base pairs?
At what cycle number does the desired double-stranded PCR product appear?
What will happen during the PCR if the annealing temperature is too low?
What happens if the extension temperature is too high?
Components of a PCR
What is a PCR ‘master mix’ & what is the advantage of using it?
What is the significance of using Taq DNA polymerase in s PCR?
What is the function of Magnesium Chloride (MgCl2) in the reaction?
The template used in your PCR reaction can either be genomic DNA (gDNA) or complementary DNA (cDNA). What is the difference between these?
Thin-walled eppendorfs are more costly to use than conventional tubes. Why do you think they are important for PCR?
Use the sequence & primer information of the gene to calculate the expected size of the PCR fragment
750 bp
What is the purpose of the DNA cleanup stage of the process?
Was your PCR successful?
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?
What is the advantage of having different restriction sites at either end of our PCR fragment?
What does DNA ligase do in the ligation reaction?
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.
What other methods of cloning are you aware of
What is meant by “competent” cells?
Why are the plates incubated upside down?
Why is the expression step important?
Cells were plated onto a plate without ampicillin. What do you expect to see on this plate tomorrow and why is this control important?
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?
Picograms to Nanograms to Micrograms
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?
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?
