Genetics

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Determining the size of a yeast DNA insert cloned into the vector pCK103 and establishing a basic restriction map.
The results of this experiment are shown below in figure 1.

Q1. Outline how the data in figure 1 would have been generated. You do not have to go into fine detail but you should demonstrate that you understand the methodology that has been used and the nature of the data.
Q2. Explain which lanes are the controls and say what important information they provide regarding the rest of the data. With clear reference to the figure, explain the pattern in each lane with reference to the plasmid, regarding the number of cuts and hence the number of restriction sites for each enzyme.
Fig 1. Gel electrophoresis of restriction digests of the plasmids pCK103and pHAM93.
Legend: NB below pCK103 is the correct name for the vector and pHAM93 the correct name for the vector and yeast insert.
The lane contents are from left to right are lane 1 – mol. size standards; lane 2 uncut pCK103; lane 3 uncut pHAM93; lane 4 pHAM93 cut with EcoRI; lane 5 pHAM93 cut with HindIII; lane 6 pHAM93 cut with XbaI; lane 7 pCK103 cut with EcoRI; lane 8 pHAM93 cut with EcoRI XbaI
Figure 2 below is a diagram of the gel in figure 1 with the numbers being the estimate of the fragment sizes in base-pairs
Figure 2: diagramatic representation of the gel in figure 1. The numbers idicate the estimated size of each fragment in base-pairs.
Legend: The lane contents are from left to right are lane 1 – mol. size standards; lane 2 uncut pCK103; lane 3 uncut pHAM93; lane 4 pHAM93 cut with EcoRI; lane 5 pHAM93 cut with HindIII; lane 6 pHAM93 cut with XbaI; lane 7 pCK103 cut with EcoRI; lane 8 pHAM93 cut with EcoRI XbaI
Q3. Explain how the gel in figure 1 was analysed to give the estimates of fragment size shown in figure 2.
Q4. The size of pCK103 is known – see the original schedule or figure 3 below – what does this tell you about the accuracy of the data? Explain.
Q5. Using the size data in figure 2 estimate the size of the yeast insert in pHAM93 showing and explaining your working.
Q6. Use the size data and the number of fragments to generate a simple restriction map for pHAM93 showing the position of the yeast insert in the vector (pCK103) and the position of sites for the restriction enzymes being tested in the experiment. The position of the sites should be in base-pairs from the “Zero point” on pCK103 (see map in the schedule, or below – figure 3).
Figure 3. Simple maps of plasmids pCK103 (the vector) and pHAM93
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Genetics

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Determining the size of a yeast DNA insert cloned into the vector pCK103 and establishing a basic restriction map.
The results of this experiment are shown below in figure 1.

Q1. Outline how the data in figure 1 would have been generated. You do not have to go into fine detail but you should demonstrate that you understand the methodology that has been used and the nature of the data.
Q2. Explain which lanes are the controls and say what important information they provide regarding the rest of the data. With clear reference to the figure, explain the pattern in each lane with reference to the plasmid, regarding the number of cuts and hence the number of restriction sites for each enzyme.
Fig 1. Gel electrophoresis of restriction digests of the plasmids pCK103and pHAM93.
Legend: NB below pCK103 is the correct name for the vector and pHAM93 the correct name for the vector and yeast insert.
The lane contents are from left to right are lane 1 – mol. size standards; lane 2 uncut pCK103; lane 3 uncut pHAM93; lane 4 pHAM93 cut with EcoRI; lane 5 pHAM93 cut with HindIII; lane 6 pHAM93 cut with XbaI; lane 7 pCK103 cut with EcoRI; lane 8 pHAM93 cut with EcoRI XbaI
Figure 2 below is a diagram of the gel in figure 1 with the numbers being the estimate of the fragment sizes in base-pairs
Figure 2: diagramatic representation of the gel in figure 1. The numbers idicate the estimated size of each fragment in base-pairs.
Legend: The lane contents are from left to right are lane 1 – mol. size standards; lane 2 uncut pCK103; lane 3 uncut pHAM93; lane 4 pHAM93 cut with EcoRI; lane 5 pHAM93 cut with HindIII; lane 6 pHAM93 cut with XbaI; lane 7 pCK103 cut with EcoRI; lane 8 pHAM93 cut with EcoRI XbaI
Q3. Explain how the gel in figure 1 was analysed to give the estimates of fragment size shown in figure 2.
Q4. The size of pCK103 is known – see the original schedule or figure 3 below – what does this tell you about the accuracy of the data? Explain.
Q5. Using the size data in figure 2 estimate the size of the yeast insert in pHAM93 showing and explaining your working.
Q6. Use the size data and the number of fragments to generate a simple restriction map for pHAM93 showing the position of the yeast insert in the vector (pCK103) and the position of sites for the restriction enzymes being tested in the experiment. The position of the sites should be in base-pairs from the “Zero point” on pCK103 (see map in the schedule, or below – figure 3).
Figure 3. Simple maps of plasmids pCK103 (the vector) and pHAM93
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