Abstract
The study focused on generating genetically modified bacteria using molecular
techniques. The study aims to insert a glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) gene from saltwater crocodile (Crocodylus porosus ) liver tissue into
Escherichia coli bacterium using various genetically modified techniques such as
RNA extraction and analysis, Reverse transcriptase – polymerase chain reaction (RT-
PCR), cDNA synthesis, Molecular cloning and transformation, plasmid DNA
extraction and purification, and gel electrophoresis. At first, RNA containing the
GAPDH gene from the crocodile liver tissue was isolated, and cDNA was synthesized
using a random primer by RT-PCR. The synthesized cDNA was cloned into a bacterial
plasmid vector using a host bacterium plasmid, and the DNA molecule was inserted.
Transformation was confirmed by screening of competent cells. Plasmid DNA was
extracted and analysed using agarose gel electrophoresis to establish the aim. As a
result, the positive control derived a sharp band of completely digest plasmid DNA
with cDNA-derived GAPDH insert. Hence, we received contamination and technical
issues as negative control had different conformation of plasmids, but the absence of
insert also serves the aim. The expected result for negative control is plasmid DNA
without the insertion. Overall, the experiments confirmed the gene's successful
insertion despite the technical issue.
Practical 1 Questions
1. Which enzymes are a major technical issue when extracting RNA? (1 mark)
RNases are enzymes that are a major technical issue during RNA extraction as they
rapidly degrade RNA and are present on human skins and surrounding laboratory
environments.
2. Hand draw and label the mechanism of autocatalytic degradation of RNA.
Include labelling of the carbon atoms of the ribose ring. (4 marks)
3. How does diethylpyrocarbonate (DEPC) increase RNA yield in this experiment?
(1 mark)
Diethylpyrocarbonate (DEPC) inactivate RNases and reduces the risk of RNA
degradation as DEPC acts on specific amino acids such as histidine, lysine, cysteine,
and tyrosine, which are in the reactive centre of RNases. By preventing RNA
degradation from RNases, DEPC increases RNA yield.
4. Which chaotropic salts are used in the ReliaPrep TM RNA tissue miniprep system?
(1 mark)
Chaotropic salts denature and break down proteins by disrupting hydrogen bonds.
Guanidine thiocyanate (GTC) and 1-Thioglycerol used in the Mini column of
ReliaPrepTM as GTC disrupts nucleoprotein complexes, releasing the RNA into
solution and isolating free of protein.
5. Name two types of hazards that are listed under the Globally Harmonised
System of Classification and Labelling of Chemicals (GHS) as being associated
with these chaotropic salts? Refer to the information in the Safety Data Sheet
(SDS). (2 marks)
The relatable hazards of chaotropic salts are Acute toxicity, Skin Irritation and Eye
Irritation under the GHS. The first aid measures in section 4 mentioned the primary
care after being in contact with hazards, such as inhaling fresh air after inhalation,
rinsing with plenty of water after eye contact, calling a doctor after swallowing,
taking off contaminated clothes or PPE and rinsing the skin with water, and consulting
a physician.
6. What is the role of the chaotropic salts in this extraction process? (3 marks)
Chaotropic salts denature and break down proteins in RNA extraction as they disrupt
hydrogen bonds and nucleoprotein complexes while releasing RNA into solution and
isolating free protein molecules such as RNases.
7. What are the advantages of snap freezing and storing biological material at -
80°C prior to use? (2 marks)
The snap freezing or freezing at -80°C preserves nucleic acids in tissues or biological
materials to prevent RNA degradation by RNases and impact the yield of the RNA
integrity. Snap freezing or Rapid freezing also prevents large ice crystals, which can
damage the biological materials.
8. After performing spectrophotometry using the Nanodrop Lite on our RNA
extraction, we added 1ul of recombinant RNAsin® ribonuclease inhibitor to the
RNA solution.
a) What was the purpose of adding RNAsin® ribonuclease inhibitor to the RNA
solution? (1 mark)
b) Would it have affected the spectrophotometry readings if we had added the
RNAsin® ribonuclease inhibitor prior to performing the spectrophotometry?
Why or why not? (2 marks)
a) RNAsin® Ribonuclease inhibitor prevents RNA degradation by inhibiting the action
of any RNases that may be present in the solution.
b) The primary role of RNAsin® Ribonuclease inhibitor is to prevent RNA degradation
by RNases. On the other hand, the wavelength absorption by protein differs from the
nucleic acid absorption, so the ribonuclease inhibitor did not interfere with the
spectrophotometry readings. Thus, adding ribonuclease inhibitor prior to
spectrophotometry readings would not affect the results but would improve RNA
integrity by preventing denaturation.
9. What would a A260/A280 ratio of 1.8 indicate about an RNA solution? (1 mark)
A ratio of approximately 1.8 is generally accepted as ‘pure’ for a DNA sample, while
a ratio of about 2.0 is regarded as ‘pure’ for an RNA sample. A260/A280 ratio of 1.8
indicates minimal protein contamination in an RNA solution.
10. An RNA extraction was performed, and a dilution made of the resulting RNA
solution by mixing 4ul of the sample with 76ul of water prior to
spectrophotometry. Spectrophotometry of the diluted solution revealed that the
A260nm value was 0.86.
a) What is the RNA concentration of the undiluted solution? (3 marks)
b) If there is 25ul of the solution remaining, what is the total amount of RNA
remaining? (1 mark)
a) RNA concentration: OD × Conversion factor × Dilution factor = 0.86 ×40×20 =
688 µg/ml = 0.688 µg/µl
b) Total volume of RNA remaining = concentration × volume = 0.688 × 25 = 17.2 µg
Practical 2 Questions
11. When running a denaturing formaldehyde agarose gel, a buffer called 10X
MOPS is commonly used in the preparation of the agarose gel and the RNA
sample. In this context, what does the 10X indicate in relation to the buffer? (2
marks)
A 10X MOPs (3-(N-Morpholino) propane sulfonic acid, 4-
Morpholinepropanesulfonic acid) buffer in the agarose gel preparation indicates 10
times concentrated stock solutions. This means that the 10X MOPs solution is 10
times more concentrated than the final working solution used to prepare the agarose
gel and the RNA sample.
12. Prior to loading RNA onto a denaturing formaldehyde agarose gel, the RNA
sample is heated to 65oC then immediately cooled on ice. What does this step
achieve and what affect does this have on the basis on which RNA molecules
migrate through the agarose? (2 marks)
Before loading RNA onto a denaturing formaldehyde agarose gel, the RNA sample is
heated to 65oC to denature the secondary structure of RNA, such as the hairpin loop
structure, which stabilizes after rapidly cooling on the ice. In gel electrophoresis,
agarose gel retards the passage of molecules based on size, and small fragments move
faster than large or complex fragments. So, RNA's hairpin structure is complex and
moves faster or slower than the linear structure, which might differ from the result of
bands.
13. What is the primary purpose of using formaldehyde in an agarose gel for
separation of RNA molecules and why is this important? What is an additional
benefit of the formaldehyde in the gel? (3 marks)
The primary purpose of formaldehyde is to retain the linear structure of the RNA by
disrupting the hydrogen bonding in RNA molecules. As the secondary structure can
affect the passage of RNA into Gel, it also alters the results. The formaldehyde also
helps to degrade RNases. So, with the help of formaldehyde, a linear RNA can be
migrated through the gel during the electrophoresis.
14. Does RNA migrate towards the positive or the negative electrode during
electrophoresis? (1 mark)
RNA is a negatively charged molecule, so it migrates towards the positive electrode
from the negative electrode under the influence of an electric current during
electrophoresis.
15. Describe what it is possible to determine regarding the purity of an RNA sample
based on visualisation of bands after denaturing agarose gel electrophoresis. (2
marks)
After denaturing agarose gel electrophoresis, we can observe the intensity of the 28S
and 18S rRNA bands and any degradation. Based on the visualization of bands, sharp
bands represent the pure and intact rRNA and smearing, or faint bands show impurity,
contamination, or degraded RNA samples.
16. Describe what you would expect to see after electrophoresis and visualisation
under UV light for a total RNA sample of high integrity (2 marks). What would
indicate a sample of low integrity? (2 marks)
After electrophoresis and visualisation under UV light, A high-quality or ‘Pure’ total
RNA will have a sharp 28S and 18S rRNA band in a 2:1 ratio. The low or inadequate
quality of RNA degraded as a smear or lack of sharp bands, and completely degraded
RNA will be seen as a low molecular weight smear, as shown in the image below.
Practical 3 Questions
17. In what type of biological entity is reverse transcriptase naturally found? (1
mark)
Retroviruses (RNA viruses) have naturally found Reverse transcriptase enzymes,
primarily responsible for converting single-stranded RNA viral genome into its
complementary DNA bases.
18. What primer strategy did we utilise in the reverse transcription reaction? Name
one other primer strategy that could have been utilised, that would still have
allowed us to successfully PCR our target crocodile gene from the cDNA
generated. (2 marks)
We use the Random primer strategy in the reverse transcription reaction. We can
successfully PCR our target crocodile gene using the generated cDNA's Oligo-dT
primer or gene-specific primer.
19. What was the purpose of heating the RNA sample to 70 oC for 5 minutes prior to
adding the other reagents? (1 mark) Why is this required prior to the reverse
transcription step? (1 mark)
The sole purpose of heating the RNA sample to 70oC for 5 minutes is to ensure the
linear RNA for PCR reaction so reverse transcriptase can bind to the appropriate
molecule for cDNA synthesis as it may be possible that RNA is in secondary or
hairpin structure, which can interrupt the binding of reverse transcriptase enzyme.
Before the heating step, ensure the presented RNA molecules are in a linear structure
for reverse transcriptase binding to cDNA synthesis.
20. Which reaction was the negative control in the PCR and what did it test for? (2
marks)
A reaction containing water instead of cDNA with a Master mix works as a negative
control. This reaction works as a check for contamination or any appearance of
amplification. If there is no amplification, indicate no contamination in PCR reagents
and surrounding environments.
21. What is the main purpose of preparing a Master Mix containing all of the
reagents required for the PCR reaction except for the DNA template, and then
aliquoting this into PCR tubes containing the relevant DNA template? (2 marks)
A master mix contains all the required PCR reagents except the DNA template, which
reduces the number of individual pipetting for each reaction, reduces pipetting errors
for minimal amounts and causes cross-contamination through the pipetting. Most
importantly, we can ensure that each PCR tube contains the same concentration and
amounts of reagents through the master mix. This is also a time-saving and reliable
step in a PCR reaction.
22. Why are thin-walled plastic tubes used for PCR? (1 mark)
Thin-walled tubes allow rapid heat conductivity and temperature equilibrium in PCR
reactions exposed to repeated heating and cooling cycles.
23. View the sequence of the Crocodylus porosus (Australian saltwater crocodile)
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in GenBank using
the accession number XM_019548942 (ncbi.nlm.nih.gov/GenBank) then enter
the accession number in the search bar at the top). Search for and location the
positions in the gene of the PCR primers we used which are given in the Practical
Manual (you will need to reverse and complete the reverse primer sequence in
order to find it in the gene sequence). Provide the nucleotide position range of
each of the primers in the gene sequence and from these calculate the size of the
PCR product that would be expected. (3 marks)
Forward Primer found from the 638 to 659
Reverse Primer found from the 884 to 903
Therefore, the length of the PCR product = 903 – 637 = 266 bps
24. How many amino acid residues are in the predicted protein sequence for
the Crocodylus porosus GADPH enzyme? You can find this information under
the GenBank entry for the gene (ncbi.nlm.nih.gov/GenBank/). (1 mark)
336 amino acid residues
25. If there were 100 copies of the GADPH gene in your cDNA template, how many
copies would there be after 35 cycles of PCR? You can assume exponential
amplification in each cycle. Show your working and give your answer in
scientific notation. (2 marks)
No. of copies produced = z × 2n = 100 × 235 = 3.44 × 1012
Practical 4 Questions
26. What was the result of each of your PCR reactions? Is this what you expected
and why? (4 marks)
The following are the expected results of PCR reactions. The bands are slightly
fainted, suggesting contamination at some point as the trace amount of DNA
contamination can be amplified. Primers appear as small and fuzzy bands as they are
small, so they run faster through the gel. cDNA product appears as the sharp bands we
amplify in this experiment. A small band of DNA PCR product suggest insignificant
amounts of contaminating DNA presence. Except for a small faint primer band, there
is no other visible amplification in the negative result, suggesting no contamination in
the PCR result.
Ne cDN
g
DNA PCR
Product
cDNA PCR
Product
Primer
s
27. What was the purpose of conducting the Wizard SV PCR Clean Up of your PCR
product and why was this important? (2 marks)
The wizard SV PCR is designed to purify PCR products from unwanted reagents such
as primers, dNTPs, chaotropic salts, Taq polymerase and other contaminants to ensure
the amplified specificity and reliability of PCR products.
28. The Wizard SV PCR Clean Up protocol suggested a 50uL elution volume
however some of us utilised a 30uL volume. What are the important
considerations when deciding on the elution volume and why might a smaller
volume be useful in this case? (2 marks)
A concentrated volume of PCR product is required for further experiments such as
ligation or sequencing. This indicates that 30ul of elusion buffer results in a higher
concentration of DNA because of the same amount of purified DNA, which might
reduce the total recovery of DNA. 50ul suggests 100% recovery of DNA, whereas
25ul suggests 98% recovery of DNA according to Wizard SV PCR clean-up protocol.
29. During the ligation step, the compatible ends of the plasmid vector and the PCR
insert molecules interact to facilitate the ligation. Describe how the compatible
overhanging ends were formed for both the vector and the PCR product. (5
marks)
The recognition site of ECOR1 is blunt, and the plasmid has a single 3’ T at the
overhanging ends. In contrast, Taq polymerase tends to add ‘A’ to the 3’ end of the
PCR product, which improves the ligation reaction's efficiency by preventing the
vector's re-circularisation.
30. The optimum temperature for most DNA ligases is 37 oC however we are
incubating our reaction at 4oC overnight. What is the reason for this? (2 marks)
The optimal incubation temperature for DNA ligase is 37°C, which may not allow the
annealing of compatible cohesive DNA overhangs. However, overnight incubation at
4°C reduces the reaction rate and increases the annealing efficiency as this ligase has
a broad range of temperatures and convenience.
31. What is the purpose of the ligation reaction which contains no PCR product?
After this ligation is transformed into bacterial cells, will the colonies which
result be blue or white when grown in the presence of ampicillin, IPTG, and X-
gal, and why? (3 marks)
The purpose of the ligation reaction without PCR product is indicated as negative
control. The negative control will grow as blue colonies in the presence of ampicillin,
IPTG and X-gal as the lacZ gene will remain intact, which allows bacteria to produce
β-galactosidase, which cleaves X-gal. In contrast, successful ligation serves as
positive white colonies in which the lacZ gene is disrupted by inserting a PCR
product.
Practicals 5 and 6 Questions
32. By what biological process will our recombinant plasmid enter the bacterial
cells? (1 mark)
Transformation
33. In the context of this practical, what does the term competent mean? (1 mark)
The bacteria with the genetic ability to accept DNA under natural or artificial
conditions are competent cells.
34. What process was used to prepare the cells to become competent? (1 mark)
Cells need to grow in log-phase to prepare the competent cells. Cells are made
permeable to DNA using artificial conditions such as chilling the cells in the presence
of divalent cations, which increase the permeability of the cell membrane to DNA.
35. If we had prepared the competent cells ourselves instead of purchasing them,
what would be the ideal positive and negative controls to ensure that the cells are
suitable for the experiment and what would each control test for? (2 marks)
If we had prepared the competent cells for ourselves, the ideal positive control would
be the competent cells with a plasmid, which are resistant to ampicillin, and the ideal
negative control would be competent cells that cannot utilize and grow on ampicillin-
containing media. The positive control cell would be viable on the ampicillin-
containing plate as confirming competent cells. In comparison, negative control
cannot grow on ampicillin-containing cells as they do not contain plasmid, as negative
control growth can be considered contamination.
36. What reagent are we using in the agar plates to select for cells that have
successfully taken up the plasmid and what is the basis of this? (2 marks)
The cells that have successfully taken up the plasmid contain a resistant gene, so they
can survive on the agar plates containing ampicillin, whereas the cell that did not take
up plasmid cannot survive on the ampicillin containing agar plates; therefore,
ampicillin is a reagent we can use for the agar plate to isolate the cells which have
taken up the plasmid.
37. Explain in detail the basis of the blue / white selection of colonies that we are
using. Include the roles of the X-gal (5-bromo-4-chloro-3-indolyl-b-D-
galactopyranoside) and IPTG (isopropyl-b-D-thiogalactoside). (6 marks)
Blue and white colonies differentiate between the colonies containing a plasmid with
PCR insert or not. The plasmid contains the lacZ gene, which produces the β-
galactosidase enzyme. X-gal is a substrate that produces blue pigment as utilised by β-
galactosidase. IPTG is an inducer of β-galactosidase expression. The blue colonies are
cells without PCR insert, so they have intact lacZ gene, producing β-galactosidase and
cleaving X-gal by producing blue-pigmented colonies. Whereas the white colonies
contain cells containing PCR inserts that disrupt the lacZ gene, there would be no
production of β-galactosidase and no cleavage of X-gal.
38. Name three scenarios that could produce blue colonies. (3 marks)
The blue colonies result from the lacZ gene intact or functional in the cell and
produce β-galactosidase and cleavage of X-gal, forming blue pigment.
If there is no PCR insert in the plasmid, it can produce the blue colonies as the lacZ
can remain intact (If the vector has re-ligated to itself)
If there is PCR insert but it is outside the lacZ, then lacZ can produce β-galactosidase
and result in blue colonies (Vector is not cut correctly)
If insert could be inserted into the vector in frame with the lacZ gene but cannot
introduce any stop codons, it may result in the production of α-peptide and result into
production of β-galactosidase and forms blue colonies
Practical 7 Questions
39. Calculate the transformation efficiency in CFU/ug if you transformed 50ul of
competent cells with 100pg of uncut plasmid, resuscitated them by adding 950ul
of SOC before plating 5ul of the cell suspension onto a plate with appropriate
antibiotic selection, which resulted in 210 colonies growing. Show your working
(3 marks)
Transformation efficiency = number of colonies / µg DNA in the transformation
procedure
= 210 / (100 / (1000/5))
= 210 / (100 / 200)
= 210 / 0.5
= 420 CFU / pg
= 4.2 × 108 CFU / µg
40. We utilised the alkaline lysis method to recover the plasmid from our
transformed E. coli cells. What is the role of each of the solutions used in this
method? Be specific with the role of individual components of the solutions (7
marks total)
There are three alkaline lysis solutions to recover the plasmid from the transformed
cells.
1. Alkaline solution 1 contains 50mM glucose, 25mM Tris-HCL and 10mM EDTA in
deionised water at pH 8.0. Glucose helps maintain osmotic pressure in the cell, and
Tris also acts as a buffer. Meanwhile, EDTA binds to divalent cations in the lipid
bilayer and destabilises the cell wall. EDTA also protects the plasmid DNA by
chelating Mg+2as, preventing bacterial nucleases from damaging the plasmid DNA.
This solution prepares cells for the lysis as it weakens the cell wall.
2. Alkaline solution 2 contains 0.2N NaOH and 1% w/v SDS in deionised water. The
SDS detergent dissolves the cell membrane and denatures cell proteins, allowing
plasmid DNA to be separated. NaOH destroys hydrogen bonds, which convert
double-stranded DNA into single-stranded DNA. As the plasmid DNA is supercoiled,
both strands cannot separate. Hence, this part of the process is known as alkaline
lysis. As in this step, cells lyse and denture DNA and proteins.
3. Alkaline solution 3 contains 5M potassium acetate and 11% v/v glacial acetic acid in
deionised water. This solution decreases alkalinity to a nearly neutral pH, which
causes the re-naturing of macromolecules. However, protein and large DNA
molecules cannot re-nature correctly, forming a large, aggregated precipitate out of
the solution. Due to decreased pH, the hydrogen bond reforms turn single-stranded
DNA into double-stranded DNA. This step is a selective procedure as the supercoiled
plasmid DNA only re-nature into double-stranded DNA. In contrast, chromosomal
single-stranded DNA is trapped within the precipitate of large macromolecules. This
step helps neutralise the solution and separate chromosomal DNA from plasmid DNA
by precipitating.
Separated plasmid DNA purified from the salts and any residual cellular debris using
a crude method for future applications.
41. Why do we invert and not vortex our tubes after adding solution II during the
plasmid preparation procedure? (2 marks)
After adding solution 2, plasmid DNA and chromosomal DNA are separated into the
solution, and our purpose is to purify plasmid DNA from the chromosomal DNA
served by inverting tubes. Vortex is the process that creates mechanical stress, which
leads to the sharing of chromosomal DNA into small fragments and contaminates the
solution. In comparison, gentle inverting ensures less mechanical stress and less
chance of breaking chromosomal DNA with minimal contamination.
42. After adding solution III a white precipitate formed. What is the precipitate
largely composed of? (1 mark)
The white precipitate primarily comprises macromolecules such as proteins,
chromosomal DNA, and other residual cellular debris.
43. What is the basis of the ethanol precipitation of our plasmid DNA and why is it
important to remove all traces of the ethanol before resuspending our plasmid
pellet in water? (2 marks)
Ethanol precipitation works by precipitating nucleic acids using ethanol and salts.
Ethanol reduces the water solubility of DNA, and salt neutralises the DNA phosphate
backbone and forms a pellet of DNA during the centrifugation process. Removing all
traces of ethanol ensured minimal contaminants of sheared chromosomal DNA, trace
nucleases and salts.
44. What is the purpose of placing your tubes in the centrifuge with their hinges
facing outwards when precipitating the DNA? (1 mark)
During centrifugation, placing tubes with their hinges facing outwards ensures that the
DNA pellet will form on the side of the tube closest to the hinge so we can quickly
locate the pellet. Sometimes, pellets are small and translucent, which can be risky if
the pellet is accidentally disturbed or lost during the removal of the supernatant.
Practical 8 Questions
45. What is the standard definition of a unit of a restriction endonuclease? (2 marks)
A unit of a restriction endonuclease means 1 unit of enzyme activity where the
amount of required enzyme produces a complete digest of 1µg of substrate DNA in a
total volume of 50µl in 60 minutes under the optimal assays’ conditions for that
restriction enzyme.
46. Star activity is a phenomenon that can occur with some restriction enzymes.
What does the term star activity refer in relation to the enzyme’s recognition
site? (1 mark) What conditions can increase the likelihood that star activity may
occur? (2 marks)
Star activity for restriction enzymes refers to the ability of certain restriction enzymes
to cleave DNA sequences that are identical but distinct from their restriction sites
when exposed to non-standard conditions.
The most commonly star activity may occur due to:
High enzyme concentration
Long reaction times
High glycerol concentration
Non-optimal reaction buffer
The presence of organic solvents
Substitution of Mg+2 with other divalent cations
47. Draw an image of the gel of your digested plasmids indicating the identity of
each of the bands in your samples and their sizes. Are the results what you were
expecting and why? (5 marks)
The following is the derived result from the experiment. The ladder bands are so
Complete digest
plasmid with cDNA
derived PCR product
Supercoiled plasmid
DNA 8000
Nicked plasmid DNA bps
3000
Linearized plasmid bps
t 1000
bps
Band of Insert
500 bps
t
250 bps
bright that the ladder was added in high volume in the gel and contaminated lane 3 as
there was no sample loaded into lane 3, and the bands were remarkably similar to the
ladder. Here, I used lane 3 to derive the band size, which may not be accurate as the
original ladder is not visible properly. Here, the contrast and brightness increased
from the original image to visualize any faint bands using the format picture option.
Lane 1 had an inoculation from the blue colonies, which shows three bands, and lane
two from the white colonies shows two bands. In lane 1, the thin-lined sharp band
may contain supercoiled plasmid DNA, which may represent Nicked plasmid DNA.
The faint band can be a linearized plasmid without insert. In lane 2, the sharp band
may be a wholly digested plasmid with a cDNA-derived PCR product. The faint band
appearing above 500 bps can be an insert. The expected result is to derive the
Insertion of PCR product in the plasmid to run between 250-300 bps and plasmid
above 3000bps in lane 2.
48. A plasmid which has been linearised with a restriction endonuclease which
cleaves the plasmid at a single point, will migrate on an agarose gel in
accordance with its molecular weight. What are the possible conformations that
a double stranded uncut plasmid can take which migrate at different rates
through an agarose gel? Indicate whether they would migrate faster or slower
than the linearised plasmid and why. (4 marks)
The factors that affect the rate of DNA molecules through an agarose gel are the size
and conformation of DNA molecules under DNA. A double-stranded uncut plasmid
can take mainly three conformations, which are supercoiled, nicked, or linearized.
Supercoiled DNA is a complex molecule with a comparably higher molecular weight
than linearized DNA, which helps it migrate faster than linearized DNA.
Nicked DNA is a circular structure, a relaxed and open molecule that moves slower
than linearized DNA due to being less compact than supercoiled DNA.
Linearized DNA is intermediate and moves according to its molecular weight.
49. What is the relationship between the percentage of agarose in an agarose gel and
the size range of the DNA fragments that will be best separated from each other?
(2 marks)
The percentage of agarose in agarose gel depends on the size of DNA fragments, as
the higher gel concentration is inversely related to the pore size of gel and DNA
fragments. The larger DNA fragments can be separated through lower agarose
concentrations as they have larger pore sizes, which allow fragments to migrate easily.
In comparison, the higher concentration of the agarose can be better for the smaller
fragments due to low pore sizes.
50. What are two roles of loading dye (also known as loading buffer) in an agarose
gel? (2 marks)
The loading dye or loading buffer has two significant roles. The colour component of
the dye works by tracking migrating DNA through the gel so we can monitor when to
stop the run. The loading dye also increases the density of the DNA sample as we can
visualise sharp bands during separation in agarose gel.
