Dissertation
Unit 13 Assignment C - Analysis of DNA
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Fully referenced and with a distinction grade :))
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Par: sarahloo711 • 4 mois de cela
Looking at the unit guidelines not all the spec has been covered in thi
Par: molliemcmillan • 4 mois de cela
Hi! It did get a distinction marked by my tutor, and there are references on it so you can add further research yourself :)) I have checked it with the brief again and it does cover the brief. Maybe you did a different practical but it is fully evaluated and explained? Would you mind telling me the parts of the spec it does not cover so Ican update
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Analysis of DNAUnit 13
Assignment C
Gel electrophoresis is the separation of DNA fragments as well as other molecules through
the negative charges of the fragments attracting towards the positive electrodes within a gel
electrophoresis machine. This is due to a current running through the gel which contains
indents of the DNA fragments. It is incredibly crucial within forensic science due to the
accuracy and ability to match offender profiles.
Theory behind restriction enzymes and DNA
A restriction enzyme is an enzyme which is produced by bacteria and has the ability to
‘cleave DNA at specific sites along a molecule’. These ‘specific sites’ are called restriction
sites, and each enzyme can only recognise a few of them. (Kara Rogers, 2019.) (1). This
means that it can cut DNA at specific areas, this produces gaps in the DNA, which contain
sticky ends. With DNA ligase, these ends can match and DNA can be joined together to form
a singular strand of DNA, meaning DNA can be altered and modified if required. They can
be ligated into each other through building a recombinant plasmid.
DNA is a polymer which is made up of various nucleotides. Within these nucleotides, a
phosphate group, a sugar molecule and a nitrogenous base is present (deoxyribose). DNA is
shaped in the form of a double helix, and holds genetic information. The bases within DNA
are as follows: adenine, guanine, cytosine and thymine. These bond together and can help
form base pairs. Base pairs can help produce unique genetic information markers, and there
are millions of them which can be analysed within a DNA sample to aid identification, which
is something that makes DNA analysis so crucial in forensic investigation.
There are also factors which can affect the separation of DNA with gel electrophoresis, for
example, if a large amount of DNA is contained within a small sized gel, it won’t be able to
migrate efficiently, potentially making results incorrect. Therefore, it is essential that the
appropriate sized gel is used for electrophoresis. As well as this, the concentration of the
agarose gel can affect the resulting resolution of the DNA fragments. (2) The time of the run
of the electrophoresis machine can also affect results, longer run times tend to create better
resolution, however may affect the band as they may end up smearing or diffusing.
Methods
Risk assessment:
Agarose gel - May cause acute health conditions, as well as weakness and drowsiness, help
hinder exposure through wearing PPE equipment, and handling with care.
Enzymes and Samples - May cause spillage risk so ensure to handle with care to not cause
spillages and slipping risks.
Boiling Water - May cause scalding burns, ensure to not cause spillages and do not directly
touch the water. Use special gloves when handling the liquid agarose gel mix.
Electrophoresis machine - Ensure no liquid is spilt on the plugs to avoid electric shock.
, DNA sample preparation -
Equipment list:
● A micropipette (2-20 microlitres)
● 4, 1.5 ml microfuge tubes.
● Floating tube rack
● Microcentrifuge
● The reagents (2.5 restriction buffer, pKAN-R, pARA, restriction enzymes and distilled
water.
1. Using the reagents as follows: restriction buffer,
pKAN-R plasmid, pARA plasmid, the restriction
enzymes BamHl and Hindlll and distilled water, the
microfuge tubes containing K+, K-, A+ and A- were
created. K+ and A+ contains the restriction
Figure 1 - The reagent list
enzymes needed for cutting into the DNA plasmids.
Whilst K- and A- do not. K- and A- will remain
undigested. These were added in the amounts as follows in Figure 1.
2. These were then spun in the microcentrifuge in order to mix the reagents together
3. The tubes were incubated in a 37 degrees celsius water bath for one hour as that is
the optimum temperature for enzyme activity.
4. This will then ensure K+ and A+ tubes have been mixed with restriction enzymes and
have now been cut open (digested by the enzymes) at the specific sites needed for
ligation.
Building a recombinant plasmid -
● Micropipette (2-20 microlitres)
● Floating tube rack
● Microcentrifuge
Figure 2 -
● 70 degrees celsius water bath
● The reagents, including: Digested pKAN-R plasmid (K+), Digested pARA plasmid
(A+), Ligation buffer (LB), DNA ligase and distilled water.
1. Place K+ and A+ tubes into the 70 degree water bath, this will allow for the restriction
enzymes to denature and stop the digestion reaction from occurring. This is done for
30 minutes.
2. A LIG tube (ligation) microfuge tube should be labelled.
3. A solution was created and placed in the LIG tube, this will allow for the ligation
(joining together) of the K+ and A+ digested reagents. The solution is shown in
Figure 2.
4. I ensured to switch between pipette tips between each pump of solution so there is
no contamination, this could be because K- and A- could end up in the digested
tubes and affect the volume of reagent and the rate of digestion.
5. We spun this tube within the microcentrifuge in order for the reagents to mix.
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