Lab practical

Question 1

DNA cloning workflow

Answer 1

Plasmids used, derived from bacterial cells.
Contain:
Replication origin
Cloning sites (MCS): sequences that are recognised restriction enzymes.
Selectable markers, usually antibiotic resistance genes.
Promoter region upstream of the multiple cloning site (expression vectors)
Polyhistidine-coding sequence (facilitate protein purification)

Question 2

Selection of positive clones

Answer 2

Bacterial cells that have incorporated a plasmid.
Incubate transformed bacteria in agar plates containing antibiotic overnight at 37 deg.

Question 3

Bacterial liquid culture

Answer 3

Inoculate a small volume of liquid agar medium containing an antibiotic with a single bacterial colony.
Grow overnight at 37deg in a shaker.
Harvest bacterial cell by centrifugation.
Discard supernatant and keep bacterial pellet.

Question 4

Miniprep of plasmid DNA

Answer 4

Used to isolate small plasmid DNA from bacteria.
Limtied contamination with proteins and genomic DNA.
Uses cell lysis and genomic DNA form a precipitate after neutralisation that is removed by centrifugation.
DNA is absorbed onto a Si matrix in the presence of high salt.
Elution from the matrix with low salt buffer.

Question 5

Plasmid States

Answer 5

Can adopt different states that are characterised by distinct migration distances.
Only the linear form can be used to reliably calculate fragment size.
Restriction digests of plasmids produced fragements with defined lengths (single vs double digest)

Question 6

Size of DNA, Calibration curve

Answer 6

Measure the migration distance from the slot to the band and plot it against known size of the fragment.
Fit a curve that described the data, display the formula in the graph, R2 value (fit quality)
Insert the migration distance of a DNA fragment of unknown into formula to calculate length.