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Restriction Digestion Samples Management

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Question:

Discuss about the Restriction Digestion Samples Management.
 
 

Answer:

Introduction:

The DNA ladder consisted of 10 bands of sizes 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 base pairs. The distance travelled by the DNA bands were measured in millimeters and a standard curve was generated. Both the DNA band sizes and the distances travelled by them were plotted to generate a standard curve. The standard curve was prepared in excel and a scatter plot was generated carrying all the respective data points. The X- axis represents the DNA length in base pairs and the Y-axis represents the distance travelled by each of the bands in the DNA ladder used. An exponential trend line was inserted in the scatter plot and the necessary equation was also included as shown in Appendix.

The unknown plasmid was digested with three different enzymes. These enzymes are BamHI, EcoRI and HindIII. The plasmid was single digested with the BamHI, EcoRI and HindIII restriction enzymes as well as done in combinations. These combinations are EcoRI and BamHI, EcoRI and HindIII, BamHI and HindIII. The undigested plasmid was also loaded in one of the lanes of the agarose gel. The DNA ladder was used to determine the sizes of the unknown bands in the lanes, where the restriction digestion samples were loaded. The distance travelled by the unknown bands were measured in millimeters. The sizes of the unknown bands were determined from the standard curve generated.

The first lane consisted of the uncut plasmid. Uncut plasmids generally consists of supercoiled and closed circular forms, so it is difficult to determine the exact sizes of the plasmid. Single digestion of the plasmid with BamHI yielded a single band of size 5.0 kb, which is the same size as the top most DNA band in the DNA ladder. Single digestion with EcoRI yielded bands of sizes approximately 3.0, 1.5 and 0.5 kb. The three bands obtained after digestion with the EcoRI enzyme coincided with the corresponding bands in the DNA ladder. Single digestion with HindIII yielded bands of sizes approximately 3.25 and 1.75, positioned between 3.0 and 3.5 kb band and between 1.5 and 2.0 kb bands of the DNA ladder, respectively. The results indicate that BamHI has a single restriction site in the plasmid, EcoRI has three restriction sites and HindIII has two restriction sites in the said plasmid.

Double digestion with EcoRI and BamHI yielded four bands of sizes approximately 2.25, 1.5, 0.625 and 0.5 kb, respectively. The 0.5 and 1.5 kb bands are common in case of both single digestion with EcoRI and on double digestion with EcoRI and BamHI. Thus, these two bands are due to EcoRI digestion only, while, the 3.0 kb band obtained in the case of EcoRI digestion was digested by BamHI to sizes 2.25 and 0.625, respectively. Thus, the BamHI site is 2.25 and 0.625 kb apart from the EcoRI sites. Double digestion with BamHI and HindIII yielded DNA bands of sizes approximately 1.75, 1.625 and 1.5 kb, respectively. Single digestion with HindIII also yielded a common DNA band of size 1.75 kb. Thus, this 1.75 kb band is caused due to HindIII digestion only, while the other two bands obtained as a result of double digestion using BamHI  and HindIII are caused as a result of digestion by BamHI. Thus, it can be inferred that the BamHI restriction site is present between the two HindIII sites and digests the 3.25 kb band obtained in the case of single digestion with HindIII to approximately 1.75 and 1.625 kb, respectively.

Double digestion with EcoRI and HindIII yielded five DNA bands of sizes approximately 2.125, 0.875, 0.75, 0.625 and 0.5 kb, respectively. Single digestion with EcoRI yielded three DNA bands of sizes 3, 1.5 and 0.5 kb, respectively. Thus, it can be seen that the 0.5 kb band is common for both the EcoRI single digestion and the EcoRI and HindIII double digestion. Thus, the 0.5 kb DNA band obtained is solely due to EcoRI digestion, while the 3 kb DNA band obtained in the case of single digestion with EcoRI underwent restriction digestion by HindIII to DNA bands of sizes approximately 2.125 and 0.875 kb, respectively. The 1.5 kb DNA band obtained in the case of single digestion of the plasmid with EcoRI was divided by restriction digestion with HindIII to DNA bands of sizes 0.875 and 0.75, respectively. Thus, there are two DNA bands of 0.875 kb co-migrating in the agarose gel. The 3.25 kb DNA band obtained in the case of single digestion with HindIII was divided into three DNA bands of sizes 2.125 kb, 0.5 kb and 0.75 kb, respectively. These three DNA bands were obtained as a result of presence of two of the three EcoRI restriction sites present between the two HindIII sites in the plasmid. The 1.75 kb band obtained as a result of single digestion of the plasmid with HindIII was divided into two 0.875 kb bands co-migrating in the agarose DNA gel. This is obtained as a result of the presence of the third EcoRI restriction enzyme site in between the two HindIII restriction enzyme sites.

 
At first single restriction enzyme digestion maps are created. The circle represents the plasmid. Figure 1 represents the presence of the single BamHI site. Single digestion resulted in the linearization of the plasmid, thereby yielding a single DNA band in the agarose gel as observed in lane 4. Figure 2 represents the EcoRI sites present in the plasmid. Single digestion with EcoRI yielded three bands as opposed to the single band obtained in the case of single digestion with BamHI and this was observed in the agarose gel in lane 3. Figure 3 represents the HindIII sites present in the plasmid DNA. There are two HindIII sites, which can be represented by the presence of two DNA bands as observed in the agarose gel in lane 5. Figure 4 represents the EcoRI and BamHI sites present in the plasmid DNA. Figure 5 represents the BamHI and HindIII sites in the plasmid DNA. Finally, Figure 6 represents the final restriction sites present in the plasmid DNA.
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