13. Synthetic Biology 2 Flashcards
What is plasmid ligation?
an enzyme-catalysed reaction which forms permanent covalent bonds between two DNA fragments.
What are ‘sticky ends’?
the two ends of the plasmid formed when both DNA fragments have been digested by the restriction enzyme.
What temperature does ligation happen at and what does this allow?
16°C - low temperatures allow hydrogen bonds to form, which hold the DNA fragment together temporarily.
What is the enzyme used in ligation?
DNA ligase
What does DNA ligase do?
catalyse the formation of new covalent bonds between the 3’ OH groups and the 5’ Phosphate groups on the DNA backbone. These are known as phosphodiester bonds.
What is the cofactor used in ligation?
ATP
What is Religation?
if the same restriction enzyme is used for the initial digest, both ends of the plasmid and insert will have complementary ‘sticky ends’
- this means that the plasmic can stick together without the insert = relegation
Why would we want to avoid using Religation?
it reduces the proportion of recombinant plasmids, thereby also reducing cloning efficiency.
What is the reagent needed to remove the 5’ phosphate groups from the plasmid?
Phosphatase
Why is the insert not treated with phosphatase?
if both the plasmid and the insert were treated, no phosphodiester could form so ligation cannot occur and cloning would fail.
- hydrogen bonds form on the sticky ends
What is other way to prevent religation?
Double digestion
What is Double digestion?
use two different enzymes for the initial digest, so that the two ‘sticky ends’ on the plasmid will be different and religation cannot occur
What does religation mean in regards to the sticky ends?
they will be the same - will have used the same enzyme.
Removal of plasmid
the small chunk of plasmid in between the cut sites will be dissociated as it is no longer attatched. a high concentration of insert increases the chances of it binding to the plasmids ‘sticky ends’.
What is the last step of plasmid ligation?
the insert will be ligated into the plasmid in the correct orientation for the promoter to drive transcription in the correct direction.
What charge is the DNA phosphate backbone?
negative
During agarose gel electrophoresis, what happens to the ions when there’s no current applied?
they diffuse outwards
During agarose gel electrophoresis, what happens to the ions when there is a current applied?
they will migrate to the positive electrode
What are agarose polymers?
spaces between the agarose fibres where DNA can travel on the gel
What is agarose gel electrophoresis?
used to separate DNA molecules by size
DNA molecules is impeded by the gel if they are…
large
What kind of molecules will show the most migration to the positive electrode?
small molecules
What is gel electrophoresis?
is the separation of charged substances through a gel using an electrical current.
Why is a buffer used?
- to keep the pH constant
- to keep the gel moist
- to provide ions for electrical conduction
How much buffer is used?
enough to cover the gel
What are the samples mixed with?
a loading buffer
what does the loading buffer contain?
- a coloured dye to track the progress while the gel is running
- a dense chemical to keep the samples In the wells
- a fluorescent dye to allow visualisation of the results
Why is a DNA marker used?
to determine the size of the DNA fragments In the samples
What does the marker contain?
a selection of DNA fragments of known lengths
What voltage is used on the power supply?
between 40 and 180 mV
How is the gel analysed?
it is placed on a transilluminator and the room light is turned off revealing the location of the DNA.
What do the number of bands indicate?
the bands correspond to the number of different sized DNA fragments present
The length of DNA vs the Bp
the shorter the DNA, the quicker the migration, the smaller the Bp.
larger molecules are delayed by the gel and so have a larger bp so will be closer to the DNA ladder.
