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Isolation & Characterisation of Proteins

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Introduction

Name: Ng Yen Pheng Student ID: 22353046 Date and day: 20 March 2012, Tuesday Title: Electrophoresis Aim: The purpose of conducting this experiment is to understand the separation technique using agarose gel electrophoresis technique. Furthermore, the experiment also aims to observe the migration of nucleic acid in the gel electrophoresis and calculate the size of DNA fragments cut by HindIII. Lastly, from the separation technique, the different sizes of a macromolecule can also be determined with the help of a marker. Result: Table 1: Fragments of DNA marker and the distance migrated (cm). Fragments size (kb) Distance migrated (cm) (Distance Migrated)-1 (cm-1) 10.0 3.4 0.294 8.0 3.8 0.263 6.0 4.2 0.238 5.0 4.6 0.217 4.0 5.0 0.200 3.5 5.3 0.189 3.0 5.8 0.172 2.5 6.4 0.156 2.0 7.0 0.143 1.5 8.1 0.123 1.0 9.5 0.105 0.75 10.7 0.093 0.5 12.2 0.082 0.25 14.3 0.070 Figure 2: Graph of (distance migrated by fragments of DNA marker)-1 against fragment size of DNA marker Table 2: Distance migrated by DNA fragments that have been digested by HindIII and their respective sizes. ...read more.

Middle

Besides that, it may also due to the staining of the gel is not sufficient to stain the other fragments, as they may be too small in size (Boyer, 2009). Difference in number of bands observed may be due to the low resolution of the gel. Some factors affecting the resolution are gel thickness, length of the gel and heat dissipation (Nakai & Modler, 1996). When the gel is thin, resolution of the gel will be higher. As for the gel length, the longer the gel, the better the separation would be. When the voltage is applied to the gel, heat is generated at the same time. When the gel is running, overheating of the gel increases diffusion of the bands. Resolution hence reduced (Nakai & Modler, 1996). From the practical, 7 fragments were found in the restriction digest at the figure 1 with the size of 25.05kb, 15.87kb, 12.19kb, 10.16kb, 7.56kb, 4.06kb and 0.04kb. ...read more.

Conclusion

In the large pores of paper electrophoresis, when voltage is applied, equally charged nucleic acids will move to the opposite pole at the same speed, without fragments being separated or only part of them will be separated. Hence, paper electrophoresis is more suitable to be used in protein molecules separation. Whereas in gel electrophoresis, the pore size of the gel can be manipulated by changing the concentration of the agarose used. Charge on the analytes, too, is one of the factors affecting the distance migrated by the sample. Because of the variable pore size, this separation method can be used to separate nucleic acids, proteins and some other molecules. Conclusion: In conclusion, agarose gel electrophoresis can be used to separate fragments which are produced by the HindIII restriction digest of ? phage DNA. The sizes of ? DNA fragments being digested by HindIII are determined by the equation from the DNA ladder graph and there were only 7 bands were visible which are 25.05kb, 15.87kb, 12.19kb, 10.16kb, 7.56kb, 4.06kb and 0.04kb. ...read more.

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