Question

Lab 8 Extension Activity: Plasmid Mapping and Restriction Enzymes Mapping the Plasmid The first step in mapping a plasmid is to determine how many times a restriction site is found on that plasmid. Examine the results for plasmid 55 as an example. The data given in the following table are for the double digest using EcoRI and Pstl. Also, giving are the data for single digests by the individual enzymes. The numbers in the columns under each enzyme represent the sizes of all the fragments that are formed when each enzyme is used for a single digest. The chart below shows the sizes of fragments that will be generated when plasmid 55 has been digested with the indicated restriction enzyme. S5 9481 bp Pul-2520 634 - quence 521 Plasmid S5 Enzymes Fragments Plasmid S5 (9481 bp) EcoRI Psti 9481 2860 2838 1986 1093 468 164 Both 2838 2817 1986 1093 468 164 72 43 Total Mapping the Plasmid Exercise.

please help me with these questions

Answer 1

1. The size of a fragment can be calculated by subtraction of the starting point from the ending point on the plasmid

Here the lambda sequence Is seen to be extending from pstl 298 to pstl 6919.

Hence size of lambda phage = 6919 - 298 = 6621 base pairs (bp)

2. Addition of the bp of fragments in each column is same as the bp of the plasmid as they are all fragments of the same Plasmid and only differ in the number and location of sites at which the plasmid is cut. All parts of the plasmid are present but fragmented. Hence they always add up to a value same as that of the plasmid

3. There is one fragment. Yes the enzyme has cut the plasmid and has resulted in a linear form of the plasmid. We can see the location of restriction site of EcoRI in the diagram Hence we know that it has cut the plasmid and linearizes it.

4. There are 7 fragments. Hence pstl has 7 restriction sites in Plasmid S5. ( Number of fragments = number of restriction sites)

5. There are 8 fragments when both enzymes are used. This indicates the presence of restriction site of EcoRI present Between the restriction sites of pstl resulting in the formation of an additional fragment . Yes, it further confirms that EcoRI cuts the plasmid.

6. Answer : Fragment of pstl 2860 bp

While comparing columns of fragments of pstl and fragments of both enzymes, we can see that fragment 2860 bp (pstl alone) is broken into two fragments of 2817 bp and 43 bp (when both enzymes used)

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