Hindil (1) 415 EcoRV ( Hindil (1) styl (2) 042 Psl ( 1017 Bamel (t) PAMP PKAN 41

Question

Hindil (1) 415 EcoRV ( Hindil (1) styl (2) 042 Psl ( 1017 Bamel (t) PAMP PKAN 4194 bp 4530 bp 11s0 Styl (2 000 Pat (2) 2137 EooRI (1) 2116 Ecol (1) 2096 Bamt (t) 384 EcoRV(1) pGLO 5371 bp 239 BamHl (3) 1465 Hindlill (2) 1510 Styl (1) 1864 BamHI (3) EcoRI (1) 3177 Pstl(22084 Bamd 102 Pstl (2) 2114 Hindil (2) Figure 1. Restriction maps for pAMP, pKAN, and pGLO, three of the plasmids found in the company stocks of bacteria. The name of the plasmid and the total number of base pairs are shown in the middle of the circle. Restriction sites are shown. The number before the restriction enzyme name is the position of the site and the number in parentheses after the restriction enzyme name is the number of times that particular enzyme cuts the plasmid. Table 1. Reagents, Tools, and Equipment Available to Perform a Restriction Digest Experiment In addition to the tools and equipment listed below, you will have access to all the necessary equipment and reagents required to analyze the results of your running buffer, a power supply, a gel electrophoresis chamber, saran wrap, and a permanent marker Liquid Reagents Tools and Equipment dH buffers Waste container for used tips Unknown plasmid DNA sample Molecular weight standard & DNA digested with FcoRI and Hindil) 6 different RE (restriction enzymes) Microfuge tube rack Microcentrifuge Crushed ice Beaker for ice BamHI EcoRV Pst . EcoRI . HindIlI Styl37C water bath

Explanation / Answer

2. The control sample would have everything you use for the reaction except for the restriction enzyme. In other words, the control sample would ideally have the unknown DNA sample, the buffer of the restriction enzyme and water in place of the restriction enzyme. It is basically a mock digestion carried out simultaneously with the actual digestion. By using this control you will know the size of the undigested DNA and hence which plasmid. It also will indicate presence of exonuclease activity in one of the components of the reaction, if no DNA band is seen in the gel after electrophoresis.

3. a. The control sample will let us know the size of the uncut plasmid DNA. From the size we will be able to know which plasmid DNA we are working with. The control sample will also indicate whether the plasmid DNA has a nick or a cut. The nick and cut (linear form of plasmid DNA) will be seen as additional bands on the gel. Intact (uncut) plasmid DNA would ideally be of the highest intensity.

b. On comparison of the digested sample to the control we will get to know approximately where the enzyme has cut the plasmid. The bands in the digested reaction would add up to the size of the control DNA. The control would also indicate the changes which can occur in the reaction independent of the restriction enzyme, for example presence of exonuclease contamination in the DNA or other reagents used in the reaction can be detected in the control sample.

4. You need a restriction enzyme which would digest the three plasmids at different number of sites so that each plasmid would have different number of bands and different band sizes. This would facilitate the identification of the plasmid been given. PstI would be ideal to use. pAMP has one restriction site for PstI, so after digestion a single band will be seen on the gel. pGLO has 2 sites for PstI hence after digestion two bands will be seen one of approximate 1000bp(3177-2102) and other approximately 4000bp. Similarly, pKAN has two restriction sites for PstI and hence after digestion, the plasmid would show two bands of ~300bp(1090-767) and ~3800bp. Hence by using PstI we will get different patterns (that is number of bands and band size) for each plasmid. And hence it would be easy to identify which plasmid was digested.

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