Isolation chelate with the metal ions in the cells.

Isolation of DNA is a very important technique which is the
foundation for many types of techniques such as the diagnosis of many genetic
diseases as well as fingerprinting DNA. How much amount and purity required,
the DNA type is what makes the difference for the different methods for DNA
isolation. There were three different E. coli cultures the aim was the analyse
the DNA of the E. coli.  Multiple amounts
of techniques were used to manipulate and isolate the DNA from E. coli. We
start of by Isolating the plasmid DNA from the 3 cultures using alkaline lysis.
Alkaline lysis is an extraction method used to isolate plasmid DNA from
bacteria.  Next the DNA which has been
isolated restriction enzymes is used for digestion. Restriction enzymes are
able to cleave DNA and make them into fragments and this is within the molecule
at sites called restriction sites. Bacterial transformation was also done. Finally,
is the analysis of the E. coli which is transformed and this is achieved by
using agarose gel electrophoresis.

Method

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Practical 1

Three E. coli cultures A, B and C were provided which had
been grown overnight shaken at 37°C. The bacterial pellet is dissolved
in 100 ml of solution 1.
Solution 1 contains 50Mm glucose the glucose aims to provide an osmotic balance
between the cell and the solution and this prevents the cells from bursting at
this stage. Solution 1 also contains 25Mm Tris (pH 8.0) this is used to
stabilise the ph in the solution.  EDTA
10Mm is also a chemical which is necessary to allow DNA degrading enzymes. The
main purpose for EDTA is to bind to magnesium and calcium and this stops the
DNA from degrading. The EDTA is also able to stabilise the DNA phosphate
backbone as well as the cell wall.

Next solution II is added, solution II is 0.2M NaOH and 1% (w/v)
SDS. This strong alkaline solution is able to disturb the cell membranes and
allows to come in contact and denature the plasmid and chromosomal DNA.   The
cell contents made contact with the extracellular chemicals which allowed EDTA
to chelate with the metal ions in the cells. SDS precipitate with proteins in
the cell contents and form insoluble complex. As a result, precipitates were
observed in the solution. Chromosomal DNA and plasmid DNA were denatured by the
high pH in the solution. The process is known as denaturation as it

Solution 3 is added next which is 3M potassium acetate pH
4.8. The potassium acetate is able to decrease the alkalinity of the solution
so it able to renature the plasmid DNA but does not renature the chromosomal
DNA. The ssDNA can re nature the dsDNA because the hydrogen bonds between the
single stranded DNA is re-established. Through hydrophobic relations a white
precipitate is formed by the SDS, denatured cellular proteins and the single
stranded genomic DNA all sticking together whereas the double stranded plasmid
dissolves in the solution.

At this point most of the cells debris is separated from the
plasmid DNA but in the solution there is the debris the salts, Rnase and EDTA
so the solution has to be cleaned up and the plasmid DNA concentrated. 70%
ethanol is added next and it is able to change the DNA’s structure as they
aggregate and precipitate from the solution. Using centrifugation, the DNA
which is precipitated can be separated.

 

 

Practical 2

From the E. coli which had been isolated next we begin to
degrade the DNA using restriction nucleases. Restriction enzymes cut DNA
molecules in specific areas to cut them into smaller fragments. Different kind
of DNA sequences are cut and recognised by different restriction enzymes. These
restriction enzymes also need a buffer which is suitable this includes
magnesium as a co factor. A certain concentration and a Tris to buffer the ph.
For different kinds of enzymes there are different optimum salt concentrations.
Samples B and C are isolated with 10 units of enzyme. There are 2 tubes called
tube BR and tube CR. Tube BR contains DNA B, 10 x EcoR1 buffer, EcoR1 enzyme
and water, Tube CR contains the same but instead of DNA B it contains DNA C.
There is a specific order in which these are added. Firstly, water is added
then it is the buffer, the DNA and then finally it is the enzyme. The reason
for this order is because a suitable environment has to be created before the
enzyme is added. Eco R1 is basically a restriction enzyme isolated from E.
coli, which at particular locations cuts DNA double helix at specific
restriction site. EcoR1 is able to make cuts in the backbone of both of the
strands, and this allows there to be two sticky ends at the cutting site of the
DNA.  There is a specific sequence which
the EcoR1 can recognise this sequence is GAATTC and the enzyme cuts in between
the G and A on the strand which is complimentary. To start the solution is
added orderly with water, buffer and the enzyme. The water is used to dilute
the buffer because the manufacturer concentrates the buffer. The EcoR1 buffer
is there as it is the optimal buffer used for the enzyme performance. When the
conditions are finally suitable for the enzyme it is added. This is when it
opens up or fragmentise the plasmid DNA. Once the both the tubes had been
completed they were incubated.

 

Transformation of plasmid

Now a technique known as bacterial transformation is used
and two tubes B and B are diluted in a Tris buffer at pH 7.6 and this makes up
40 folds of the final volume of the mixture, as they have been diluted they are
labelled diluted B and diluted BR. The purpose of bacterial transformation is
to introduce DNA into bacterial cells. There are many techniques used to
achieve this but the reliable technique is a heat shock technique. When the DNA
has been taken up it has to either join with a host genome or autonomously
replicate. Circular forms of DNA are the only DNA which are going to be able to
replicate, the linear form which use restriction enzymes will not be able to
transform. The circular form when introduced to E. coli will be able to
replicate.

 

The heat shock technique uses calcium chloride which creates
a calcium rich environment, between the plasmid DNA and the bacterial cellular
membrane the rich calcium environment cancels the electrostatic repulsion
between them. In the bacteria pores are created as there was been a sudden
increase in the temperature so this allows the entry of plasmid DNA to the
bacterial cell.  When the cell takes up
the DNA it establishes itself to create a steady tranformant. In the practical
the unknown strain of E.coli cells were added with calcium chloride and pre
cooled in ice. The procedure is repeated twice and kept in ice. At the same
time tube 1 which contains no plasmid DNA is prepared, tube 2 containing
diluted plasmid B is prepared and tube 3 containing diluted plasmid BR is
prepared. The pre cold competent cells were added to the tubes 1 2 and 3 and
mixed gently. As the cells are fragile, it is important to avoid using the
vortex. After 15 minutes the tubes were shocked with hot water at 42°C. At this
stage the cell membrane becomes thinner and plasmid DNA can enter the cell
body. After 2 minutes the tubes were set in ice for 5 minutes to allow the cell
membrane to recover. L broth is added to each tube and water bathed at 37°C for
at least 20 minutes. After that cells in each tube were transferred LB amp
plate spread and incubated overnight.  

 Practical 3

Agarose Gel electrophoresis is a common used method for
analysing the size, purity, quantity and the sequence of DNA molecules and
plasmid DNA molecules. Agarose is a polysaccharide it is a one of the
components to agar and is extracted from red seaweed. It is also made up of anhydrous-galactose
units. There are many reasons why the agarose gel is beneficial for gel
electrophoresis. Between the polysaccharide unit’s non-covalent bonds are
formed by the agarose gel. A sol state is formed as non-covalent bonds hold the
structure of the agarose gel so it undergoes a phase transition at high temperature,
When the running buffer and the agarose powder is mixed it creates the gel with
later arrangement of the sol state at a higher temperature and also arranged
cooling.

To start an agarose gel is prepared by the molten agarose
being poured in the former. Wells are formed in the DNA sample for the DNA to
be loaded by combs, they are then left for approximately 20 minutes to set.
Once the gel has been set TBE buffer is used to carry a current and provide
ions it is also able to maintain the Ph.

As we know DNA is negatively charged, so when the electric
field is applied for the period of electrophoresis there will be movement of
the DNA towards the anode which is the positive pole. The sample loading wells
be towards the negative pole which is the cathode, so when the gel is placed in
the electrophoresis tank it is orientated.

A loading buffer solution is used to treat the plasmid
sample before it is loaded on the gel. 
The density is increased of the sample as the loading buffer contains
glycerol.  DNA is able to travel towards
the positive electrode as the larger fragments are slowed down in compare to
the smaller fragments which is why they do not travel far. A band is also
formed as all the fragments gather at a point and travel at more or less the
same rate. So now when all the fragments have travelled and separated the different
sized fragments, there is a dye known as SYBER-SAFE and this is used to
visualise the DNA.  When the dye is hit
by a UV radiation there is an orange coloured fluorescent light.  Finally, the UV trans illumination
photographs the gel which contains the stained DNA molecules.  A loading buffer completes the circuit as well
as balances the pH in the gel.

Results

The transformed E. coli from tube 1, 2 and 3 were grown in
the agar plate. Tube contained 0 colonies, tube 2 contained 300 colonies and
tube 3 had contained 5 colonies. Tube 1 contained 0 colonies as it only
contained buffer. Tube 2 had contained 300 colonies as it contained circular
plasmid DNA. Tube 3 had only contained 5 colonies as it only contained linear
DNA so the only way it may have contained colonies may have been contamination
or mutation.

From the results Tube C shows it did not travel far as the
molecules were large or had a low molecular charge so they were not able pass
through the gel network. Incomplete precipitation of the chromosomal DNA could
be a possible error.

It is known that tube A contains no plasmid but tubes B and
C contain plasmid. It is because the results show that there are 2 DNA bands on
both tubes B and C but there are no DNA bands in tube A. The plasmid DNA which
is within E. coli is in a circular form. 
The bands on the top of tubes B and C are known as nick DNA they were
linearized by alkaline solutions and are not able to renature. Circular DNA is
able to travel further as its shape causes less friction and it is smaller in
size. The nick DNA does not travel far as it is in a linear form which causes
more friction. And it has a larger molecular size.

Plasmid DNA in tube C is cleaved into two fragments by the
restriction enzyme because it contains two recognition sequences. The band of
the two fragments can travel further than the band of plasmid DNA C because
they experience less resistance. Therefore, the band of the linear plasmid DNA
C has lower position than circular plasmid DNA C. Whereas plasmid in tube B
cleaves at only one recognition site the circular plasmid opens up forming a
nick DNA. Which experience higher friction than the circular plasmid DNA. Therefore,
circular plasmid DNA C can travel faster than the nick plasmid DNA C forming a
band at a low position.

The marker X contains linear double stranded DNA with no
molecular weight. When it is loaded in electrophoresis, the DNA with different
molecular weight runs to different position and forms a DNA ladder. By
comparing the bands of linear plasmid DNA from B and C with the ladder the
molecular weight of the plasmids is known. 
Linear plasmid DNA of E. Coli B has a molecular weight of 3kbp. The
linear plasmid DNA fragments have molecular weight of 4.7 and 5.3 kbp therefore
the molecular weight of plasmid DNA C is 10 kbp

Discussion and Conclusion

To start most of the macro molecules such as the proteins,
chromosomal DNA and high molecular weight RNA were all removed by the process
of Alkaline Lysis. But from the results it has shown there was some
macromolecules which had remained in tube C. The possible reason for the Tube C
containing the macromolecules could be Tube B+ had the RNA degraded using the
RNase.  When the alkaline lysis
experiment was performed there was no single stranded linear DNA when
denaturing was done, and this has shown that there was control on the pH. 5
colonies of antibiotic resistant were grown on plate 3 by linearized plasmid B.
There was linearized plasmid in tube BR but at low points the reason for this
is there was no circular plasmid DNA seen in electrophoresis. Post inspection
should be done on the colonies formed on the L-amp plate.  When bacterial transformation had been carried
out there may have been a chance some of the plasmid B which is linearized has
merged with the chromosomal DNA.