Bacterial plasmids experiment Flashcards
What do you treat E. Coli with so you can transform them?
Calcium chloride
What fraction of cells take up the DNA and what are they called?
1/10000, competent
When a DNA molecule is denatured by heating what happens?
H bonds between complementary strands of double helix broken but covalent linkages in backbone stay intact
What happens when a linear piece of DNA is denatured by heating?
2 strands become completely separated
Meanwhile what happens when a small covalently closed molecule is heated?
Intertwining of the 2 strands means that complementary strands remain close to each other
When linear denatured DNA molecules are allowed to renature quickly what happens?
What happens to small plasmids?
What does this mean?
Linear –> Insoluble network that precipitates forms
Plasmids –> remain in soluble phase as they renature efficiently
Means plasmids will remain in the soluble phase can then be removed by precipitation with ethanol
What were the major aims of our plasmid experiment?
1) To transform a strain of E. Coli with a mixed preparation of plasmid DNA
2) To identify the plasmids on the basis of phenotype and size
What 2 antibiotic resistance genes did we use in our plasmid experiment?
Ampicilin and chloramphenicol
How did we make a control?
Don’t add plasmid DNA prep to one of the cell suspensions in calcium chloride
How did we vary temperature of E. Coli to transform them?
1) cells left on ice for 20 mins after prep added
2) Cells heat-shocked by adding to 42 degree heating block for 2 minutes
What agar plates did we spread the transformed cells onto to obtain colonies?
Ampicillin and chloramphenicol plates
If colonies are obtained implies genes coding for antibiotics carries on plasmids have been taken up
What unusual appearance was seen in some of the colonies on the ampicillin plate?
Darkly coloured, shows mel+ genotype
Fact only found on amp plate indicated amp R gene and mel+ gene carried on same plasmid
How did we separate plasmids on basis of size?
Run PAGE
What ladder did we run alongside our samples?
Supercoiled molecular ladder, used to create standard curve for distance migrated in gel against molecular weight, plot on LOG paper
(see notes for diagram)
In gel elecrtophoresis why did extra bands form in each lane?
Due to DNA being present in different combinations
