Biosciences BB1804 Part 3: Molecular Analysis Assessment Details

This assignment, Part 3 of the BB1804 Molecular Analysis assessment, focuses on the analysis of experimental results from a practical involving PCR, restriction enzyme digests, and agarose gel electrophoresis. Students are required to analyze a provided gel image showing expected results, particularly for those who failed to generate the expected digestion pattern. The core of the assignment involves calculating the sizes of PCR products and DNA fragments generated after restriction enzyme digestion. This is achieved by drawing a graph of distance traveled versus log10 DNA size using data from Hyperladder I. Students must complete a table with Hyperladder I data, construct a graph in Excel, and then use the graph to calculate the sizes of DNA fragments in base pairs, providing calculations and explanations. Finally, the assignment requires completion of a table showing the calculated sizes of all DNA fragments present on the gel from the Part 2 experiment.

Part 3
For evaluation of the Part 2 results, all the students who failed to generate an expected
digestion pattern of the wild type DNA and mutant DNA should analyse the gel showing the
expected results and provided by the supervisor (file: Part 2 gel example). The analysed gel
should be shown in Figure 2B of the BB1804 assessment Part 2.
Table 1
To calculate the size of the PCR products (non-cut samples in figure 2) and DNA fragments
generated after the restriction enzyme digestion (cut samples in figure 2) in the Part 2
experiment, you need to draw a graph of distance travelled from the well versus log10 DNA
size of Hyperladder I. To do this you need to print the image of the gel, measure distance
travelled by each band of HyperLadder I from the well, and complete Table 1:
Size of DNA (bp)
in Hyperladder I
Log10 Size of
DNA
Distance (mm) travelled
from the well
2500 3.40 1.9
2000 3.30 2.2
1500 3.18 2.5
1000 3.0 3.0
800 2.90 3.3
600 2.78 3.7
400 2.60 4.2
200 2.30 4.9
PCR Products
I
Unknown
2.3
II 2.3
III 2.3
IV 2.9
V 3.5
VI 2.3
VII 2.3
VIII 2.9

IX 3.5

Figure 3
Use the data from the Table 1 and Excel Program to build the graph of distance travelled
from the well versus log10 DNA size of the DNA molecular weight markers as shown below.
Insert your Graph here:
2.2 2.4 2.6 2.8 3 3.2 3.4 3.6
0
1
2
3
4
5
6
Distance (mm) travelled from the
well
Linear (Distance (mm) travelled
from the well )
Distance (mm) travelled from the well
Log10 Size of DNA
Calculations of the sizes of the DNA fragments generated by PCR and restriction
enzyme digestion
Using this Graph and knowing the distance travelled by the DNA fragments generated by
restriction digestion, calculate the sizes of these DNAs in base-pairs (bp). Provide the
example of calculations of the sizes of DNA fragments with a brief explanation how it was
done. Word limit – 50.
Insert your calculations and explanations here:
Slope = -0.36 (By SLOPE () function in excel)
y = mx + C
For known x and y (i.e. x = 1.9, y = 3.4), C = 4.08
For x=2.3mm, y=3.27, Antilog103.25 = 1778bp
For x=2.9mm, y=3.04, Antilog103.04 = 1096bp
For x=3.5mm, y=2.83, Antilog102.83 = 676bp

Table 2
Complete Table 2 below to show the calculated sizes in base-pairs (bp) of all the DNA
fragments generated by PCR and restriction enzyme digestion that are present on gel from
the Part 2 Experiment (Figure 2):
DNA
Enzyme
DNA 1
Fragments in bp
DNA 2
Fragments in bp
DNA 3
Fragments in bp
Non-cut
PCR 1778bp 1778bp 1778bp
Nhe I
digested
fragments
1778bp 1096bp, 662bp 1778bp, 1096bp,
676bp