Here are my answers to part 1 & 2 can you give me the answer to 3& 4 1. A) yes,

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Here are my answers to part 1 & 2 can you give me the answer to 3& 4

1. A) yes, the image of gel electrophoresis clearly is showing two bands in sample section which represents the supercoiled and relaxed form of plasmid the first column represents the ladder that is used as standard and also to reduce the size of the sample. There should be a third band representing the linear form of a plasmid, but as the plasmid sample was uncut, it will not have its linear form.

B) To know whether your plasmid size, we compare the bands obtained in sample column to the ladder used in electrophoresis. Now among the two bands of plasmid, we will use the higher one as it is the relaxed form of the plasmid as it will give a much closer image of its size. Generally, the ladder used in such experiments for plasmid its size ranges from 0.5 kb to 10kb /1Mb in both the cases the plasmid we obtained is of expected size as the expected size of a plasmid in its relaxed state is from 2kb to 1Mb

2. the control is that sample which remains untreated with experimental conditions. i.e., control sample will only contain untreated sample plus buffer but no reagents to ensure that whatever results that we will obtain are because of the reagents/condition we used to treat that initial sample. we keep every condition same except one condition whose difference we are trying to test.

for restriction digestion of plasmid we want to understand the effect of restriction enzyme on plasmid, therefore, we treat our plasmid with restriction enzymes and then pour them into the agarose gel and the control used in this experiment is the untreated plasmid and this sample will contain everything same as a treated sample except the enzyme,(i.e., buffer +water +untreated plasmid DNA+ tracking dye) by doing this we get to know the actual plasmid DNA integrity.

To complete this assignment, read the document and answer the questions posed. Your answers must be TYPED in Lab Archives. You must include the questions as well as the answers. (If you handwrite your math instead of typing it, you may photograph and upload the photo to Lab Archives as an attachment.) Your company maintains stocks of different strains of bacteria that it uses to produce enzymes and proteins. Unfortunately, the technician you replaced did a poor job keeping track of these stocks and now it is not clear which is which. You have been given the task of determining the identity of some unknown stocks. Different strains of bacteria can potentially be distinguished from one another by looking for differences in the plasmids they contain. One method for accomplishing this is by Restriction Fragment Length Polymorphism (RFLP) Analysis. In RFLP Analysis, DNA samples are cut into fragments using Restriction Enzymes. Because they have different DNA sequences, plasmids can be distinguished from one another by digesting them with the same enzymes and comparing the number and sizes of the DNA fragments produced. On the next page are restriction maps for three of the plasmids found in the company stocks of bacteria (Figure 1). Your supervisor gave you a culture of one of these strains and you have already performed a miniprep (during the Molecular Biology-II lab) to isolate the plasmid DNA from this strain. Now your job is to design a restriction digest experiment that will enable you to determine which plasmid the bacterial strain contains.

Explanation / Answer

3)

a) What information will it provide regarding your plasmid DNA?

The control sample will provide

1) information regarding the size of the plasmid

2) Intactness of the plasmid (ie., the appearance of two bands indicate that the plasmid is intact. If we see more bands in control plasmid means the plasmid is degraded)

b) Why will it be useful to compare the control sample with your digest reaction?

After the digest reaction, the plasmid gives multiple bands. The size of all the bands when added should be equal to the size of the plasmid in the control sample.

4) Which enzymes will you use to determine the identity of the plasmid found in the bacterial strain

I will use HindIII and BamHI to digest the plasmids

Upon digestion, pAMP plasmid will give the digested fragments of two sizes - 784 bp (7.84 kb) and 3755 bp (3.7 kb).

Upon digestion, the pKan plasmid will give the digested fragments of two sizes - 1861 bp (1.8 kb) and 2333 bp (2.3 kb)

Upon digestion, the pGLO plasmid will give the digested fragments of four sizes - 226 bp, 399 bp, 250 bp and 875 bp.

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