Cutting DNA with restriction enzymes a partial digest
Restriction enzymes are produced naturally by bacteria and cut the DNA at specific sequences. The sequences that are recognized are usually inverted repeats (palindromic) and the cuts that are made on the DNA are double stranded symmetrical cuts. Cleavage produces either cohesive or blunt ends. The mode of action of these enzymes is that they hydrolyze the phosphodiester bonds at specific cleavage sites present in the recognition sequences in each sugar-phosphate backbone of the DNA strand (Black, 2005 Pierce, 2003 Barnum, 1998 Stryer, 2002).
In partial digestion, the restriction enzymes are allowed to act on the DNA sequences for only a limited time. The objective is to prevent the enzyme from cleaving all the available restriction sites in the DNA. This generates large overlapping fragments or contigs that can be cloned using suitable vectors. Separation of the DNA fragments on the basis of their size differences can thereafter be carried out using gel electrophoresis (Pierce, 2003).
This experiment sought to partially digest the pRcCMV plasmid using BamH1 by varying the reaction times. BamH1 is an example of a type II restriction enzyme and it is isolated from Bacillus amyloliquefaciens. The recognition sequence for this enzyme is 3-CGATCC-5 and it produces cohesive ends. The pRcCMV plasmid is 5542 bp long and has 3 BamH1 restriction sites and these are situated at 909 bp, 1306 bp and 3350 bp respectively (Pierce, 2003). Since the DNA is circular, cleavage of the plasmid using BamH1 was expected to yield 3 fragments for each of the test reactions. The sizes of the fragments were expected to be 397 bp (1306-909), 2044 bp (3350-1306) and 2192 bp (5542-3350) respectively. The control was expected to yield one fragment since no cleavage occurs as the plasmid DNA was not exposed to the enzyme. The band representing the fragment produced was expected to be located close to the loading point due to its relatively big size.
Materials and methods
The BamH1 restriction enzyme was used to cleave the plasmid DNA in reactions that were performed at 37c. The reactions were carried out in 4 different tubes, one of which was the control tube. The reaction buffer, the plasmid DNA and enzyme were added to all the test tubes. No enzyme was added to the control tube. The reactions in the 3 test tubes were timed and were allowed to proceed for 2, 10 and 30 minutes respectively. GlycerolEDTA was used to stop the reactions. The loading dye was added into each of the 4 tubes, the gel prepared and the samples loaded into separate wells. The gel was then placed into the tank and run for 5-7 minutes at 275V. The bands obtained were then photographed.
Difficulties encountered during the experiment included leakage of the gel mixture and that the gel had speckles after destaining had been done. Leakage of the gel mixture was stopped by correctly aligning the spacers and the bottom of the glass plates. To get rid of the speckles, the stain was filtered and this removed the precipitates present which were the cause of the speckles.
In partial digestion, the restriction enzymes are allowed to act on the DNA sequences for only a limited time. The objective is to prevent the enzyme from cleaving all the available restriction sites in the DNA. This generates large overlapping fragments or contigs that can be cloned using suitable vectors. Separation of the DNA fragments on the basis of their size differences can thereafter be carried out using gel electrophoresis (Pierce, 2003).
This experiment sought to partially digest the pRcCMV plasmid using BamH1 by varying the reaction times. BamH1 is an example of a type II restriction enzyme and it is isolated from Bacillus amyloliquefaciens. The recognition sequence for this enzyme is 3-CGATCC-5 and it produces cohesive ends. The pRcCMV plasmid is 5542 bp long and has 3 BamH1 restriction sites and these are situated at 909 bp, 1306 bp and 3350 bp respectively (Pierce, 2003). Since the DNA is circular, cleavage of the plasmid using BamH1 was expected to yield 3 fragments for each of the test reactions. The sizes of the fragments were expected to be 397 bp (1306-909), 2044 bp (3350-1306) and 2192 bp (5542-3350) respectively. The control was expected to yield one fragment since no cleavage occurs as the plasmid DNA was not exposed to the enzyme. The band representing the fragment produced was expected to be located close to the loading point due to its relatively big size.
Materials and methods
The BamH1 restriction enzyme was used to cleave the plasmid DNA in reactions that were performed at 37c. The reactions were carried out in 4 different tubes, one of which was the control tube. The reaction buffer, the plasmid DNA and enzyme were added to all the test tubes. No enzyme was added to the control tube. The reactions in the 3 test tubes were timed and were allowed to proceed for 2, 10 and 30 minutes respectively. GlycerolEDTA was used to stop the reactions. The loading dye was added into each of the 4 tubes, the gel prepared and the samples loaded into separate wells. The gel was then placed into the tank and run for 5-7 minutes at 275V. The bands obtained were then photographed.
Difficulties encountered during the experiment included leakage of the gel mixture and that the gel had speckles after destaining had been done. Leakage of the gel mixture was stopped by correctly aligning the spacers and the bottom of the glass plates. To get rid of the speckles, the stain was filtered and this removed the precipitates present which were the cause of the speckles.
Categories:
1 коммент.:
Creative Enzymes holds its unique position in the enzyme market to provide strategic solutions for finding promising industrial enzymes from various natural sources, such as soil microbes, plants and animals. discovery of enzymes
Отправить комментарий