Introduce single plasmids into recipient bacterial cells
To then allow the cells to replicate in order to achieve high yields of plasmid DNA
The cells chosen for this experiment are DH5α E.Coli competent cells. Competent cells are specially adapted so that they have the ability to adopt foreign DNA. They have been treated to become permeable to small DNA molecules.
Cooling the cells on ice for 5 minutes prepares them to become permeable to foreign DNA and the sudden heat-shock at 42oC causes the DNA to pass into the cell.
The DH5α strain has been chosen as it has been made deficient in some genes, therefore protects the inserted DNA.
LB growth medium is added in the experiment as it contains nutrients such as sodium chloride and yeast extract which assists cell growth. This helps maximise the yields of plasmid DNA.
Plasmid Prep:
The aim of the plasmid prep experiment is to:
Grow up a starter culture of bacterial cells
Extract the plasmids from the cells
Purify the DNA
Ampicillin is an antibiotic and is added to the starter culture mixture to kill off any unwanted bacteria that has invaded the solution. The inserted plasmids have an ampicillin resistance gene, so only the transformed cells are able to survive in the presence of ampicillin.
Centrifuging the starter cultures forces all the cells to the bottom of the falcon tube into a ‘pellet.’ This means all the unwanted solution can be discarded.
Cell lysis buffer is added to break open the cells so that the DNA is released. It is important to isolate the DNA as the following experiments (digestion and ligation) modify the DNA strands and hence require direct access to the plasmids.
When the reaction mixture is centrifuged for the second time, all the ‘empty’ cells will be forced to the bottom of the falcon tube and the DNA will be suspended in solution. Hence, the solution is poured into a column so that the DNA can be caught and be subsequently washed.
PCR (Polymerase Chain Reaction):
The aim of a Polymerase Chain Reaction is to:
Instigate DNA replication to increase DNA concentration.
Stage 1: Denaturing
When the PCR experiment is prepared, the DNA component is in the plasmid form, hence it is double stranded. However, DNA replication can only occur on single stranded DNA therefore the first step in the reaction must be to separate the two strands. This is known as Denaturing and occurs when the reaction is subjected to very high temperatures.
Stage 2: Annealing
DNA replication occurs when an enzyme called Polymerase attaches itself to the DNA strand and copies all of the base-pairs. However, Polymerase cannot bind to single-stranded DNA therefore small segments of DNA called Primers are added to the reaction. They attach themselves to the base-pairs at the beginning and end of the DNA strand, creating two areas of double stranded DNA sandwiching an area of single stranded DNA. This process is called Annealing and creates an area that Polymerase can bind to which allows DNA replication to begin.
Gotaq buffer and MgSO4 are important in this step as they support the polymerase enzyme to ensure that it maintains its shape. If the enzymes shape is altered (by a varying pH) then it cannot bind to the DNA strand.
Stage 3: Extend
The Polymerase enzyme copies all the base pairs and creates new single strands of DNA.
Digestion:
The aim of Digestion is to:
Cut DNA strands to either remove a sequence of base pairs or open up a plasmid.
The digestion experiment is undertaken in either a 20ul or 50ul total volume reactions. The vector DNA (the ‘opened’ plasmid into which the insert is introduced) would not have undergone a PCR reaction hence will have a lower concentration than the insert. Therefore, it will be undertaken in a 50ul volume reaction to try to increase the quantity of vector DNA.
The restriction enzymes used depends on which restriction sites need to be cut. When the reaction finishes, the mixture is heated to 65oC to denature the enzyme.
The DNA fragments are purified to make sure the required fragments are isolated, as the enzymes don’t always cut the intended restriction sites and therefore lead to unwanted DNA strands.
Gel Electrophoresis:
The aim of Gel Electrophoresis is to:
Identify the DNA molecules present
When the gel is placed in the electrophoresis tray, it is positioned so that the loading wells are located next to the negative electrode. This is because DNA is negatively charged; hence when the DNA is loaded into the wells and the system is subjected to an electrical current the samples will run towards the positive electrode. If the wells were positioned near the positive electrode, the samples would run off the gel into the TAE buffer.
The distance the DNA travels is dependant on the number of base pairs the sample contains. The smaller the DNA strand, the further it will travel down the gel.
Ethidium Bromide is added to the gel to make the DNA samples visible under Ultra-violet light
Ligation:
The aim of Ligation is to:
Join two samples of DNA together
The vector and insert components are added in a 3:1 ratio respectively. As the vector did not undergo PCR it is necessary to increase the quantity of the vector to ensure that insert-vector ligations occur.
Sometimes during ligation, the vector fragments fuse back together to reform complete plasmids without binding to an insert. Therefore, when the ligation is finished and the mixture is plated to grow up colonies, some of those colonies will not be the ligated product but the original plasmids. Therefore, a ‘Vector Alone’ control is prepared. As no insert was added to the control, only reformed plasmids will grow on the control agar plates. If the biobrick transformation plate has far more colonies than the vector alone plate, there is a good probability that some of those colonies will be ligation products. This gives an indication that the ligation has been successful.
The reaction mixtures are subjected to 65oC heat in order to denature the ligase enzyme.
The buffer is present to support the ligase enzyme and ensure that it maintains its shape.
"
