Hi, I\'m missing question 4 and 5 and I\'m confused about it and i don\'t really

Question

Hi, I'm missing question 4 and 5 and I'm confused about it and i don't really know the answer, can someone help me please? I'll attach my Lab notes along with the lab manual 10.

Thank you

Week 10 - Restriction Enzyme Post-Lab Questions

1- In today’s lab, you purified plasmid DNA from E. coli. What types of molecules are present in the E. coli that you started with?

Proteins, RNA, and DNA are the major biomolecules that are being separated during DNA isolation.

2- After the purification what types of molecules do you expect to have in your sample?

DNA is expected to be present in sample.

3- What restriction enzyme did you use for the digest today?

Specific name of restriction enzyme is not mentioned in this protocol so more information is needed.

4- What size(s) of DNA fragments do you expect to get from the restriction enzyme digest if your bacteria contain the plasmid with no insert?

5- What size(s) of DNA fragments do you expect to get from the restriction enzyme digest if your bacteria contain the plasmid with the gene of interest?

6- Based on what you know about how restriction enzymes work explain why you are able to predict the sizes of DNA that will be obtained from this experiment.

DNA Module Week 10 - DNA Miniprep Introduction In lab today you will be given a pellet of E. coli bacteria. These bacteria contain one of the two plasmids whose sequences you analyzed last week. The goal of this module is to determine which plasmid is in your bacteria. Today you will isolate the plasmid DNA from the bacteria and will set up a restriction enzyme digest to cut the DNA. Background The purification strategy for plasmid DNA, and for any biomolecule for that matter, is to get rid of as many contaminants as possible and to isolate the desired product free of other cellular contaminants. The major contaminants in this purification are cell membranes, RNA, chromosomal DNA and E.coli proteins. We will use a commercially available kit to remove the contaminants and to obtain purified DNA. This kit is manufactured and sold by Qiagen. Restriction enzymes are bacterial enzymes that cleave DNA at specific sites based on unique base pair recognition sequences. This ability of restriction enzymes to recognize specific sequences of DNA makes them useful in the lab. Different restriction enzymes recognize and cut at different DNA sequences (see Figure 1). This property allows us to predict where in a DNA sequence (such as a plasmid) a particular restriction enzyme will cut the DNA. In last week's lab you analyzed the DNA sequence of the plasmid and the plasmid with the gene of interest in order to determine how they would be cut by particular restriction enzymes. In today's lab you will use restriction enzymes to cut the plasmid DNA you are going to purify. In the following weeks we will determine the sizes of the produced DNA fragments and will use this information to determine whether or not your plasmid contains the gene of interest. 87

Explanation / Answer

4) Here as there is no insert we can get only the plasmid DNA as a result of the restriction. The size of the DNA fragment is equal to the size of the plasmid.

5) Here we would obtain two fragments upon restriction digestion, one is the gene of interest and the other fragment is the plasmid DNA. Provided that the Plasmid DNA do not contain any other restriction sites in other parts of the plasmid molecule, except at the region called multiple cloning sites.The plasmid DNA is larger in size than the gene of interest.

As the size of both Plasmid DNA and gene of interest is not mentioned in the question, We cannot say about the exact size of them.

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