Lab test Flashcards
What should happen when plating competent cells on LB agar
Tests cell viability of competent cells
Lawn of cells should be evident
What should happen when you plate competent cells on LB agar containing ampicillin
Checking for no intrisnic resistance for ampicillin
Should have no cells
What should happen when you plate competent cells plus a super coiled plasmid with amp resistance on LB - agar containing ampicillin
Competent cells should transform and distinct colonies will occur on plate
Why must plasmid DNA be supercoiled in order to transform E.Coli
Replication must occur to provide sufficient expression of Amp gene in order to confer resistance to ampicillin. Transformation requires replication and replication requires supercoiled DNA
Why are restriction endonucleases stored in glycerol?
Storing in glycerol prevents enzyme from freezing
and avoids freezing/thaw cycle which may damage the enzyme
How does EDTA stop restriction endonuclease reaction?
Chelates to Mg2+ required for enzyme activity
Why is bromophenol blue added to the loading dye
To visualise and track progress of electropheresis
Why does the loading dye contain sucrose?
For density, helps sample sink into the wells.
When extracting DNA from a blood sample, explain what happens when the
blood is mixed with the hypotonic cell lysis solution. Where is the DNA after
this step?
Red cells takes up the fluid from hypotonic cell lysis solution and the red blood cells burst. The DNA remains in the white blood cells as white blood cells have a stronger membrane
When extracting DNA from a blood sample, explain what happens to the
protein/DNA complex (chromatin) when it is mixed with the nuclei lysis solution which is a solution containing high salt (~2.5 M NaCl). Where is the DNA after this step?
The high salt solution disrupts protein/dna interactions because these interactions are weak and non-covalent. The salt disrupts the electrostatic interactions and H-bonds
DNA is released from chromatin and will be in solution.
Why does a plasmid that is partially digested give rise to many bacterial clones?
Because the plasmid is only partially digested there is remaining covalently closed circle and these can replicate once inside E.coli.
Replication required for transformation and cccDNA is required for replication.
What happens when you plate competent cells on agar that doesnt contain ampicillin
To check competent cells viability
Explain why there are 6 potential reading frames produced when a cDNA sequence is translated using computer software.
DNA has two strands. Both strands are analysed. Each codon is 3 nucleotides, therefore 3 potential start sites. 2 x 3 = 6.
How would you select the open reading frame from the six potential reading frames
It would need to begin with a start codon and end with a stop codon, with no
nonsense codons in between
What do the bands in the lanes labeled PCR 1 and 2 represent? Explain your answer.
PCR1 = Amplification of the insert in plasmid 1 using primers that flank the
MCS. Recombinant plasmid, amplicon contains insert.
PCR 2 = Amplification of the MCS only. No insert so amplificon is small.
