Emily Bullas – Genetic Engineering
1. Practical Report:
This practical used the hot shot DNA extraction method to extract DNA from a cheek cell sample.
This method uses 4 steps – cell harvesting, cell lysis, protein digestion and DNA precipitation. Once
DNA has been extracted, PCR and gel electrophoresis will be used to amplify part of the TAS2R38
gene, which contains a C/T mutation which contributes to taste perception. People with the CC and
CT mutations can taste biterness, while people with the TT mutation cannot. This will be used to
determine whether the samples can taste bitterness or not.
The equipment needed for this practical include salt water, microcentrifuge tubes, transfer pipette,
pipette tips, micropipette, bento lab set, 2 small cups, 1% agarose gel and 0.5x TBE buffer, DNA
ladder and loading dye. It also requires an alkaline lysis buffer, neutralisation buffer, a mix of four
primers – 2 to copy the whole fragment and 2 to copy either the C or T mutations – and a
mastermix.
To carry out the DNA extraction, a sample was first taken by rinsing the cheeks with a saltwater
solution to collect some cheek cells. This solution was then spat into a cup and 1.5ml was pipetted
into a labelled microcentrifuge tube. The microcentrifuge tube was placed into the centrifuge,
making sure that it was balanced correctly for safety. The sample was then spun at 4000 G for 90
seconds. After this, the sample has separated into the pellet (1), a small white ball of cheek cells at
the bottom of the tube, and the supernatant (2), a clear liquid on top of the pellet which is poured
away.
Figure 1: Pellet and Supernatant
75ul of alkaline lysis buffer is then added to the tube to break open
the membranes of the cells and nuclei. The pellet is resuspended by
flicking the bottom of the tube. The sample is then transferred into a
0.2ml PCR tube, filled as much as possible with the sample. The
sample is labelled and loaded into the thermocycler which is used to
heat the sample at 95°C for 30 minutes. After heating, the sample is
cooled for a few minutes and then a 75ul of neutralisation buffer is added to the tube. The sample is
then diluted 1:10 by adding 2ul of DNA sample to 19ul of PCR grade water, in a new PCR tube.
To carry out the PCR stage, first 4ul of mastermix is transferred into an empty PCR tube, along with
2ul of primer mix. Then, 4ul of DNA template is added to the tube and the sample is repeatedly
heated and cooled in a thermocycler to facilitate DNA amplification. The thermocycler carries out 35
repeats of a 3-step cycle – 30s at 95°C, 30s at 56°C, 30s at 72°C. This is followed by 120 seconds at
72°C.
The final stage of the practical is gel electrophoresis. To begin, 5ul of loading dye is added to the
sample to stain the DNA, as well as add weight to it so that it can sink into the wells of the agarose
gel. 5ul of DNA ladder (for comparison) is pipetted into the first well, followed by 5ul of the DNA
sample in the second well, using a fresh pipette tip to avoid contamination. Once the samples have
been loaded and the location of each sample on the gel is noted, the lid can be put on and the leads
connected, before running the gel for 40 minutes at 50V. After it is completed, the gel can be
visualised by placing the gel box onto the Bento lab transilluminator surface with a bright blue light
shining through. Using an orange filter to observe the gel, bands of light should be seen where the
DNA has separated. Figure 2 shows the results obtained.
, Figure 2: Gel Electrophoresis
The results show that the two samples tested did travel through the gel but did not separate as
expected. The DNA ladder shown in figure 2 displays the separation that was expected, with smaller
DNA molecules travelling further across the gel. Since the samples did not separate, it was not
possible to analyse the results as shown in figure 3 below. It should also be noted that the DNA
ladder cannot clearly be interpreted as a rip in the gel obstructs the view, so it would be difficult to
analyse the results even if the samples had separated.
Figure 3: Interpretation of Gel Electrophoresis
Figure 3 shows the guidelines for interpreting the results of gel electrophoresis. This was not
possible for the samples tested in this practical, so the subjects’ abilities to taste bitterness cannot
be determined.
1. Practical Report:
This practical used the hot shot DNA extraction method to extract DNA from a cheek cell sample.
This method uses 4 steps – cell harvesting, cell lysis, protein digestion and DNA precipitation. Once
DNA has been extracted, PCR and gel electrophoresis will be used to amplify part of the TAS2R38
gene, which contains a C/T mutation which contributes to taste perception. People with the CC and
CT mutations can taste biterness, while people with the TT mutation cannot. This will be used to
determine whether the samples can taste bitterness or not.
The equipment needed for this practical include salt water, microcentrifuge tubes, transfer pipette,
pipette tips, micropipette, bento lab set, 2 small cups, 1% agarose gel and 0.5x TBE buffer, DNA
ladder and loading dye. It also requires an alkaline lysis buffer, neutralisation buffer, a mix of four
primers – 2 to copy the whole fragment and 2 to copy either the C or T mutations – and a
mastermix.
To carry out the DNA extraction, a sample was first taken by rinsing the cheeks with a saltwater
solution to collect some cheek cells. This solution was then spat into a cup and 1.5ml was pipetted
into a labelled microcentrifuge tube. The microcentrifuge tube was placed into the centrifuge,
making sure that it was balanced correctly for safety. The sample was then spun at 4000 G for 90
seconds. After this, the sample has separated into the pellet (1), a small white ball of cheek cells at
the bottom of the tube, and the supernatant (2), a clear liquid on top of the pellet which is poured
away.
Figure 1: Pellet and Supernatant
75ul of alkaline lysis buffer is then added to the tube to break open
the membranes of the cells and nuclei. The pellet is resuspended by
flicking the bottom of the tube. The sample is then transferred into a
0.2ml PCR tube, filled as much as possible with the sample. The
sample is labelled and loaded into the thermocycler which is used to
heat the sample at 95°C for 30 minutes. After heating, the sample is
cooled for a few minutes and then a 75ul of neutralisation buffer is added to the tube. The sample is
then diluted 1:10 by adding 2ul of DNA sample to 19ul of PCR grade water, in a new PCR tube.
To carry out the PCR stage, first 4ul of mastermix is transferred into an empty PCR tube, along with
2ul of primer mix. Then, 4ul of DNA template is added to the tube and the sample is repeatedly
heated and cooled in a thermocycler to facilitate DNA amplification. The thermocycler carries out 35
repeats of a 3-step cycle – 30s at 95°C, 30s at 56°C, 30s at 72°C. This is followed by 120 seconds at
72°C.
The final stage of the practical is gel electrophoresis. To begin, 5ul of loading dye is added to the
sample to stain the DNA, as well as add weight to it so that it can sink into the wells of the agarose
gel. 5ul of DNA ladder (for comparison) is pipetted into the first well, followed by 5ul of the DNA
sample in the second well, using a fresh pipette tip to avoid contamination. Once the samples have
been loaded and the location of each sample on the gel is noted, the lid can be put on and the leads
connected, before running the gel for 40 minutes at 50V. After it is completed, the gel can be
visualised by placing the gel box onto the Bento lab transilluminator surface with a bright blue light
shining through. Using an orange filter to observe the gel, bands of light should be seen where the
DNA has separated. Figure 2 shows the results obtained.
, Figure 2: Gel Electrophoresis
The results show that the two samples tested did travel through the gel but did not separate as
expected. The DNA ladder shown in figure 2 displays the separation that was expected, with smaller
DNA molecules travelling further across the gel. Since the samples did not separate, it was not
possible to analyse the results as shown in figure 3 below. It should also be noted that the DNA
ladder cannot clearly be interpreted as a rip in the gel obstructs the view, so it would be difficult to
analyse the results even if the samples had separated.
Figure 3: Interpretation of Gel Electrophoresis
Figure 3 shows the guidelines for interpreting the results of gel electrophoresis. This was not
possible for the samples tested in this practical, so the subjects’ abilities to taste bitterness cannot
be determined.
