RestrictionEnzyme Laboratory Report
RestrictionEnzyme Laboratory Report
BackgroundInformation
Restriction enzymes found in bacteria. They are DNA cutting enzymesand have found significant use in genetic research. The DNA cuttingenzymes are used in sequencing work for their precision. Restrictionenzymes recognize and cut DNA at particular sequences of nucleotides(Jones et al., 2016). Enzymes “scan” the DNA molecule whilelooking at specific sequences. The sequences comprise four to sixnucleotides. Upon recognition of the sequence, the enzyme cuts thestrands from both sides but by running in opposite directions. Theprocess of cutting results in the production of blunt or sticky endsdepending on how the particular enzyme conducted the same. Unevencutting results in the production of the sticky ends while cutting ineven manner results in the formation of blunt ends of nucleotides(Schuler, & Zielinski, 2012).
In this experiment, an in vitro analysis of DNA molecules would beconducted by the use of recombinant DNA techniques such as genecloning, plasmid extraction, and restriction enzyme analysis. Theexperiment will be carried out in a procedural manner from ligationcontrol and ligation reaction, the transformation of the recombinantDNA into E. coli, culturing of kanR Transformants, extraction ofrecombinant plasmid DNA to the analysis of restriction enzymes.
Objectives
Understand the concepts of restriction digest
Understand the use of micropipette, agarose gel, and electrophoresis
Compare the DNA bands on a gel to those on a restriction map
Understand significance of restriction enzymes
Methodology
LigationControl and Ligation Reaction
Microcentrifuge tubes were labeled as C1 and R1 where C1 containedthe ligation control gel sample while the R1 contained the ligationreaction gel sample. A fresh pipette tip was used to transfer 20μlof the stock ligation reaction mixture to tube C1. The tubecontaining T4 DNA Ligase was centrifuged to collect the enzyme at thebottom of the tube. Next, 40 μl of the stock ligation reaction wasadded to the tube containing T4 DNA Ligase. The solution was thenmixed by pipetting, and the ligation reaction was collected at thebottom of the tube. Samples were incubated at room temperature forfive minutes. Samples were then incubated in a water bath for 30minutes at a temperature of 16˚C. 20μl was transferred from thesample to reaction tube R1. Next was the addition of 5μl gel loadingsolution was added to both the tube serving as a control (C1) and thereaction tube (R1).
Ligation of Plasmid Vector to kanR Gene Fragment
The plasmid vector was prepared by the digestion by a restrictionenzyme that was compatible with the foreign DNA. The plasmid vectoris mixed with the DNA sequence of the kanR gene in the presence ofthe T4 DNA ligase (Loenen, Dryden, Raleigh, Wilson, & Murray,2014). The DNA ligase allows the formation of phosphodiester bondsbetween the 5’-phosphate and the 3’-hydroxyl resulting in acovalent bond. The kanR Gene fragment formed a bond with the plasmidvector. The reaction was conducted at a temperature of 16 ˚C forefficient ligation process.
Transformation of Recombinant DNA into E. coli
The ligation reaction was diluted by mixing adding five μl of theDNA derived from T4 DNA Ligase tube with 45 μl of pure water in aMicrocentrifuge tube. The tube was labeled as a “Diluted LigationReaction”. The two tubes for control and reaction are labeled C2and R2 respectively. 500 μl of Calcium chloride was transferred tothe C2 tube by use of a sterile pipette and placed on ice. Next, fiveisolated colonies of E. coli plate were transferred to the C2 tubeand resuspended. 250 μl of the cell suspension were transferred tothe R2 tube and placed on ice. Ten μl of control DNA was added to C2and put on ice. Similarly, ten μl of diluted ligation reaction tothe R2 and put on the ice. Both samples were gently mixed andincubated on ice for ten minutes. The transformation tubes were putin a water bath for ninety seconds at a temperature of 42˚C. Theheat shock step allowed for entry of the DNA into E.coli.
The tubes were then returned to the ice and incubated for twominutes. It was followed by a transfer of 250 μl of the LuriaRecovery Broth to the tubes and mixed by flicking the tubes. Thecells were then incubated for ten minutes at a temperature of 37˚Cin a water bath to allow for the cell walls to express the antibioticresistance gene. The cells were allowed to recover and tubes labeled“Control” and “Ligation.” The cells were removed from thewater bath and centrifuged for five minutes at full speed. The 0.4mlof the supernatant was removed and discarded from the C2 tube andresuspended in 0.1ml of the remaining liquid. All of the recoveredcells from C2 tube were spread to a plate by use of a sterileinoculating loop and covered. The steps were similarly repeated forthe ligation cells in the R2 tube with a fresh pipette. The cellsuspension was given time to be absorbed by the agar plate. Theplates were then placed in an overnight incubation for 37˚C in abacterial incubation oven. The results were analyzed through thedetermination of the transformation efficiency.
Culturing of the kanR Transformants
A culture tube was picked, and an inoculated with isolated coloniesfrom the tube labeled ligation. The tube was then placed in a 37˚Cshaking water bath and cultured overnight.
Extraction of Recombinant Plasmid DNA
A Microcentrifuge tube containing the pelleted E. coli cells waslabeled. 200 μl of TEG buffer was added to the bacterial pellet andresuspended by pipetting up and down. The cell suspension was thenincubated for five minutes at room temperature. Next, 5V of RNase wasadded to the cell suspension and mixed followed by addition of 350 μlof lysis buffer and gently mixed. 200v of Potassium Acetate solutionwas then added to the suspension and mixed thoroughly until a whiteprecipitate formed. The sample was then incubated for five followedby centrifugation for another five minutes. The supernatant was thentransferred to a 1.5ml Microcentrifuge and the tube containing thepellet discarded. Next, the 0.6 ml of isopropanol was added to thesupernatant followed by incubation for ten minutes at roomtemperature.
Restriction Enzyme Analysis
For 1.5ml Microcentrifuge tubes were labeled as 3, 4, 5 and 6. 150 μlof pure water, 25 μl restriction reaction buffer, and 25vresuspended recombinant plasmid was mixed in a Microcentrifuge. Thisformed the restriction digest cocktail. 40 μl of restriction digestwas added to each of the labeled tubes. Ten μl of pure water wasadded to tube three that served as restriction digest control. Fiveμl of pure water and five μl of EcoRI enzyme was added to tubefour. The same was done for tube five containing the Pvu11 enzyme andClal enzyme to tube six. Tapping of the tubes then mixed therestriction digestion reaction.
The samples were then incubated for 37˚C for one hour followed byaddition of 5 μl of 10* gel loading solution to each of the reactiontubes. The samples were then electrophoresed.
Results and Discussion
Ideal products of ligation derived were the plasmid containing thefragment of foreign DNA. The successful transformation of thecompetent E. coli cells is inefficient by the use of the linearmolecules (Clark, 2013). Because of the same, it could be mandatoryto have the optimization of the circular molecules that contain themultiple arrays of the vectors. Also, the large recombinant moleculesthat contain multiple arrays of the vector and the insert may not beable to replicate efficiently (Stevens, Cheng, Li, Xie, Hong, Maire,& Wang, 2013). Treatment with alkaline phosphatase may be prudentin other instances.
Conclusion
Restriction enzymes have found significant use in the field ofmolecular biology research. There are different types of restrictionenzymes that have been classified depending on the regions that theycut their DNA substrates. The restriction enzymes operate by makingincisions at two points of the particular DNA substrate. The enzymesare found in archaea and bacteria. They are useful for their abilityto provide defense against viruses. They execute the tasks by cuttingup any foreign DNA while the host DNA is protected from modificationby an enzyme named methyltransferase. The enzymes form therestriction modification system. Much of the enzymes are availablecommercially and are used for different research purposes. Notably,they are used in molecular cloning.
References
Clark, M. S. (Ed.). (2013). Plant molecular biology—a laboratorymanual. Springer Science & Business Media.
Loenen, W. A., Dryden, D. T., Raleigh, E. A., Wilson, G. G., &Murray, N. E. (2014). Highlights of the DNA cutters: a short historyof the restriction enzymes. Nucleic acids research, 42(1),3-19.
Jones, J. A., Vernacchio, V. R., Sinkoe, A. L., Collins, S. M.,Ibrahim, M. H., Lachance, D. M., … & Koffas, M. A. (2016).Experimental and computational optimization of an Escherichia colico-culture for the efficient production of flavonoids. Metabolicengineering, 35, 55-63.
Schuler, M. A., & Zielinski, R. E. (2012). Methods in plantmolecular biology. Academic Press.
Stevens, M., Cheng, J. B., Li, D., Xie, M., Hong, C., Maire, C. L.,… & Wang, T. (2013). Estimating absolute methylation levels atsingle-CpG resolution from methylation enrichment and restrictionenzyme sequencing methods. Genome research, 23(9),1541-1553.
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