Plasmids and Ligation Process - Lab Report Example

?Practical Aim: To perform plasmid miniprep to isolate pBlueSkript KS II from E.coli and to perform double restriction digestion on pProEx with the enzymes BamHI and Hind III to release the FtsZ insert. Secondly, to increase the efficiency of ligation by alkaline phosphatase treatment of the linearised plasmid. Introduction: Plasmids are extrachromosomal DNA present in the bacterial species. They are double stranded DNA which forms circles with size ranging from 1 kb to 200kb ( kilobase ). Plasmids are very advantageous for the genetic engineering. Plasmids code for many antibiotic regions and they have the ability to accept the gene of interest. The transformation of our gene of interest into the plasmid is called recombination and the bacteria are called recombinant bacteria. Thus plasmids can be used as cloning vehicles or vectors. The first step of transformation is the isolation ofhte plasmid DNA from the given bacteria culture. The basic method or DNA isolation is 1. Cutluring the host cell containing the Plasmid DNA. 2. Harvesting and lysing the cell to separate the DNA from the cell organelles. 3. Separation of chromosomal DNA and Plasmid DNA through precipitation method. 4. Plasmid DNA isolation and purification. Since both chromosomal and plasmid DNA will remain in the solution, the method to isolate plasmid DNA from the Chromosomal DNA is precipitation method. Larger DNA molecules (i.e. chromosomal DNA), bound to the proteins are separated from the Plasmid DNA when the protein is precipitated. The plasmid DNA which remains in the solution is then precipitated using ethanol. Method: 1. A single colony of Bacteria containing the pBlueSkript KS II was grown overnight in the Luria Betroth overnight with ampicillin as the antibiotic. 2. From the overnight culture, 1.5 ml of the culture was taken in the centrifuge tube and centrifuged at maximum speed for 1 minute. 3. The supernatant containing the medium is discarded and the cell pellet was kept as dry as possible. 4. The cells were resuspended in the 100µl of GTE buffer and mixed gently using the pipette to ensure that no cell pellets remain in the solution. 5. To the cell pellets, 200 µl of cell lysis buffer was added at room temperature. The tube was mixed gently by inverting the tube up and down five times and incubated at ice for 5 minutes. 6. To the mixture 150 µl neutralization buffer was added and again inverted gently up and down 5-6 times. 7. The mixture was centrifuged at maximum speed for 10 minutes and the supernatant was added to the new tube. 8. To the supernatant, 1000 µl of 100% ethanol was added to precipitate the DNA. 9. The tube is centrifuged for 10 minutes in maximum speed. 10. The supernatant was removed from the tube and to the whitish DNA pellet, 1ml of 70% ethanol was added and the tube was inverted several times and centrifuged at maximum speed for 2 minutes. 11. The supernatant was removed from the solution and to the DNA, 500 µl of 70% ethanol was added as final wash. The tube was again centrifuged at top speed for 2 minutes and the DNA pellet was obtained. 12. The pellet was resuspended in 40 µl of 10mM Tris- HCl with RNase. The tube was mixed by flicking the tube and incubated at 37° C for 5 minutes. 13. 5 µl of the Plasmid DNA was transferred to sterile microfuge tube and was labeled as B3- 5 µl PKS II- southern blot and stored at -20°C. Result and discussion: The DNA was extracted from the culture using the miniprep method. The plasmid DNA obtained in this method is used for the transformation process. Answer 1: Ampicillin is an antibiotic that resists the growth of the ampicillin senstitive strains when added to the medium. As our plasmid PKS II codes for ampicillin gene, ampicillin was induced in the growth medium to avoid contaminants. Answer 2: RNase is the enzyme that cleaves the RNA present in the given sample. RNA are the contaminants seen along with the plasmid DNA. Hence RNase was added to cleave the RNA. Answer 3: We can use alkaline lysis/ phenolic extraction method or alkaline lysis/PEG method for isolating plasmids from the protein and cell debris apart from the traditional alkaline lysis/acetate method. Practical 1B: Aim: To digest the pProEX and pBKS II plasmid with BamHI and HindIII restriction endonuclease enzymes for the excision of Pc-ftsZ gene from the pProEX plasmid and to prepare the pBKSII plasmid to receive the Pc-ftsZ gene for ligation. Introduction: Restriction endonucleases are the bacterial enzymes that cleaves the double stranded DBA at specific ends. Restriction endonuleases are of many types. Tpye II restriction endonuleases are mainly used for the DNA cleavage at specific sites. Restriction endonucleases are present in many strains of bacteria and they are specific for each strain. These restriction endonucleases digest the foreign DNAs at specifc ends and thus safe-guarding the parent strain. These Restriction endonucleases require specific temperature, pH for maximum activity. The restriction enzymes are stored at 4°C to maintain their activity level.S Methods: 1. Two separate tubes were taken and the restriction digestion reactions were carried out in each tube as follows: PBKSII diegestion: pProEX + FtsZ digestion 1.3µl of 10X buffer 2 1.3µl of 10X buffer 2 2.10µl pBKSII DNA 2.15µl pProEX DNA 3.11µl dH2O 3. 6µl dH2O 4.3µl BamHI 4.3µl BamHI 5. 3µl HindIII 5. 3µl HindIII 2. The mixture was kept at 37°C for 45 minutes. 3. The mixture was then incubated at 65°C for 10 minutes to stop the reaction. Result and discussion: The mixture was kept at 37°C , which is the optimum reaction temperature for restriction reaction. After the reaction is over, the mixture is kept at higher temperature to stop the reaction. A1: Since there are only one restriction site for both BamHI and Hind III restriction endonucleases, two fragments will be formed. The fragment size will be 29 bp and 2971bp respectively. A2: The recognition sequences for BamHI is G?GATCC , EcoRI is G?AATTC, Kpnl is GGTAC?C and Sacl is GAGCT?C. (Roberts and Murray 1976). Practical 1C: Aim: To treat the BamHI / HindIII double digested PBKS II plasmid with alkaline phosphatase. Introduction: Alkaline phosphatase is the enzyme that rmoves the phosphate group form the 5’ terminals of the DNA or RNA. This is the most important restriction enzyme that is used for the cloning of the DNA. After the restriction digestion, there is greater chance for the DNA molecules to self ligate. This is prevented by using the alkaline phosphatase enzyme. Method: 1. 2.0µl of alkaline phosphatase enzyme is added along with the 3.5µl of 10x AP buffer to the 30µl of the BamHI/Hind III double digested pBKS II DNA and incubated at 37°c for 1 hour. 2. The mixture is then heat inactivated for 10 minutes at 65°C and ice cooled and stored for further use. Result: The above figure shows the restriction digestion of the BamHI / HindIII double digested PBKS II plasmid. Discussion: The lane 3 carries the BamHI / HindIII double digested PBKS II plasmid. There are two thick bands in the gel which indicates that the DNA was cut properly by the two restriction enzymes. Similarly in the lane 5, the cloning vector was also cut into two fragments by the same restriction enzymes which indicate that the digestion was complete. Practical 2: Aim: To separate the DNA fragments in the given sample by using the Agarose gel electrophoresis and to check whether the digestion was successful and to separate and purify the DNA fragments for ligation. Introduction: Agarose gel Electrophoresis is the standard method for the separation of the DNA fragments based on their molecular weight. DNA fragments move through the Agarose gel under the influence of the electrical power. As DNA is negatively charged, it moves towards the anode. The pore size of the Agarose separates the DNA fragments based on their size. Thus the DNA fragments get separated based on molecular size, the current applied, concentration of Agarose and the conformation of the DNA. A standard ladder is used to identify the size of the fragments. The Bands on the gel are visualized using the intercalating, fluorescent dye, Ethidium bromide. When the Agarose gel dyed with EtBr is visualized in the ultra violet rays ( UV rays), we can observe the bands. This method is very efficient as it can detect the dsDNA concentration as low as 1 nanogram. Method: Agarose Gel Preparation: 1.1% Agarose gel was prepared by mixing 1 gram of Agarose in 1XTAE buffer and boiling it until it completely dissolves in the buffer. 2. Ethidium Bromide of concentration 0.5µg/ ml was added to the gel at 50°C. and the gel was poured into the gel casting tray and the comb was inserted into it. The gel is allowed to solidify for 30 minutes at Room temperature. Sample preparation: 3. 6µl of 6X gel loading buffer was added to the 35 µl of pBKS II plasmid digest and mixed gently. 4. 5µl of 6X gel loading buffer was added to the 30 µl of Pc-ftsZ insert and mixed gently. 5. The gel was kept in the electrophoresis tank and 1X TAE buffer was added to the tank until the gel is slightly covered. 6. To the first lane 5µl of molecular marker was added. 7. To the second lane 20µl of pBKS II plasmid digest was added and to the third lane 20 µl of Pc-ftsZ insert was added. 8. To the fourth and fifth lane undigested pBKS II DNA and pProEX DNA were added by the demonstrator. 9. The Electrophoresis set up was run at 110 V for 50 minutes. After that the power is turned off and the gel was removed from the tray and kept in the plastic container. Practical 2B: Aim: To observe the bands of Restricted DNA fragments and to excise the required bands for purification and ligation. Introduction: The Ethidium Bromide is a florescent dye that gets illuminated under the UV light. When UV light of wavelengths 260, 300 and 360 nm are used, the DNA bands can be observed very efficiently almost to the concentration of 1 nanogram. Ethidium Bromide binds to the intercalating planar group between the base pairs of nucleic acid. The selected bands are excised from the gel and the DNA fragment is made to bind to the spin column containing silica based filters. To the silica based filters, the DNA binds and the contaminants are washed away. The DNA from the membrane is eluted using the microbiology double grade water or suitable buffer. Method: 1. Two sterile cups were obtained and the empty weight of the tube is determined. The tubes were labeled as plasmid ( pBKS II) and insert (pProEX). 2. The gel was kept in the UV box using gloves. 3. Under the UV light, the required DNA fragment is sliced without disturbing the entire gel. 4. The slice was then transferred to the labeled microcentrifuge tube. 5. The weight of the gel slice in the tube is measured for future analysis. 6. To the gel slice, add 3 volumes of QG buffer and incubate it at 50°C for 10 minutes. The tube was mixed by inverting it 2 or 3 time. 7. After the gel was completely dissolved, 700µl of the sample was added to the spin column allowing the DNA to get bound o the membrane very efficiently. 8. The spin column was centrifuged at 10000 rpm for 1 minute and the filtrate was discarded. The procedure was repeated for 2 or 3 times and the concentration of the DNA bound to the membrane was increased. 9. To elute the sample bound to the membrane, 700µl of the Wash buffer was added to the spin column and centrifuged at 10000 rpm for 1 minute. 10. The spin column was kept in the clean sterile micro centrifuge tube and 30µl of the EB buffer was added to the membrane and again centrifuged for 10000 rpm for 1 minute. 11. The DNA thus obtained was stored at -20°C for further use. Results and Discussion: After solubilizing the gel, iso propanol was not added to the mixture. Isopropanol precipitates the DNA present in the sample. As this step was missed in the experiment, the DNA was not able to precipitate and separate from the sample. This error produced impure DNA sample along with cell debris and RNA fragments. Practical 2C: Aim: To ligate the two DNA fragments for to obtain a recombinant plasmid. Introduction: Two DNA fragments are ligated using the Bacteriophage T4 DNA ligase. T4 DNA ligase catalyses the binding of 3’hydroxyl end of one nucleotide and 5’ phosphate end of other nucleotide, using the phosphate- di- ester bond. T4 DNA ligase will ligate the blunt ends, overhanging and complementary and sticky ends. It is easier to stick the sticky ends than the blunt ends. This catalytic reaction requires Mg2+ and ATP as the cofactors. The amount of the Ligase enzyme required varies based on the nature of the filaments to be ligated, stability of the hydrogen bonds to be ligated, and temperature and concentration of the DNA fragments. Method: 1. In a clean test tube, the plasmid and the insert were added as follows: Purified linear pBKS II plasmid DNA – 4 µl. Purified insert – Pc-ftsZ product - 12 µl. T4 DNA ligase - 2 µl. 10x ligation buffer - 2 µl. 2. Incubate the mixture at 20 minutes. 3. The tubes were placed at -20°C for further use. Result : The above figure is the Agarose gel electrophoresis for the ligation reaction. The lane 1 contains the marker, and the other lanes contain the ligated mixture. Discussion: Ligation is the process of joining together two DNA fragments. The ends were sticky ends and hence the ligation was very quick and perfect. The components were of equimolar and the ligation reaction was good. The insert and the plasmid DNA were borrowed from the other group and instructor for ligation reaction. The efficiency of the ligation can be checked using the Agarose gel electrophoresis. The ligation is perfect as can be seen from the Agarose gel electrophoresis. The ligation has occurred and this is confirmed by the thick single band present in the gel. A1: Ethidium bromide can be used both in the gel and in the buffer for the identification of the DNA. When added in the gel, as Ethidium bromide being positively charged will move out of the DNA and only less amount of Ethidium bromide will be found bound to the DNA. Similarly the regions where the ethidium bromide has not migrated out of the gel will create high intense background when viewed under the UV light. In this procedure a large amount of EtBr gets wasted. In the post-electrophoresis method, 0.5µg/ml concentration of EtBr is prepared in the buffer and the gel is submerged in the staining solution for 15 minutes. This method reduces the EtBr waste to a large extend as the solution is stable for a maximum of 2 months. The bands become visible from the top and bottom part of the gel and high contrast results can be obtained by using this method. (Boyer 2000). A2: Submarine gel electrophoresis is derived from the pulse-field gel electrophoresis system. In submarine gel electrophoresis method, uniform electric field is applied across the gel and lane to lane comparision is very easy. The gel lies inside the buffer, hence the chance for gel drying is negligible. For shorter DNA fragments, the resolution is very high in submarine gel electrophoresis. (Westermeier 2006). A3: Undigested pBKS II and undigested pProEX DNA were added into the gel to check whether restriction has occurred in the given sample or not. If restriction has occurred in the given sample then we can observe different bands in the gel when compared to a single band in the undigested samples. If not, it confirms that there is some error. A4: ligation of sticky ends is more efficient than the ligation of the blunt ends. The compatible sticky ends join easily with the complement pair through hydrogen bonding. This creates a relatively stable structure for the enzyme to work on. The sticky end ligation bond is stable for a long time when compared to the blunt ends enabling the enzyme to act upon very easily. The phospho- di- ester bonds are also formed very quickly in the sticky ends. In blunt end ligation, the enzyme must wait for the association of the two ends by some bonds. Blunt end ligations are usually carried out at high DNA concentrations only. Generally the blunt ends are converted into sticky ends for increasing the efficiency of ligation. (Siwach and Singh 2007). Practical 3: Aim: To perform heat-shock transformation of Competent E.coli cells with ligated recombinant DNA and selecting the colonies using ampicillin resistance. Introduction: E.coli is the main hosts for Bacterial research as it can uptake foreign plasmid DNA more efficiently than other bacteria. E.coli and its plasmid structure is well understood many other bacteria. This is the main reason for using E.coli for transformation. This uptake of the plasmid DNA is achieved at the mid log phase of the bacterial growth. E.coli is sensitive to ampicillin, and this helps us to select the colonies form the non-tranformed bacterial colonies. pBKS II contains a multiple cloning site with lac Z gene, origin of replication and ampicillin resistance region. The cloning of the gene of interest is performed at the multiple cloning site and hence the bacteria containing this plasmid will be resistant to ampicillin. The lacZ gene codes for the enzyme ?- galactosidase. This is a hydrolase enzyme that hydrolyses the ?- galactosides into mono saccharides. ?- galactosidase is activated in the presence of the lactose when the glucose concentration is low.this is the most common reporter gene or marker gene in gene expressions and transformations. ?- galactosidase blue / white screening is the base for recombinant clones. If the cloning occurs in the ?- galactosidase gene or lac operon, the production of the lac Z? is disrupted, the cells shows no ?- galactosidase activity. The presence or absence of ?- galactosidase is detected by the X- gal, a modified galactose sugar, which produces characteristic blue color when cleaved by this enzyme. For the proper activation of the gene, an inducer called IPTG ( Isopropylthio- ?- D- galactosidase) is used. Multiple cloning sites are present in the pBKS II plasmid. This region also contains the lac operon. If our gene of interest is inserted peoperly into the pBKS II plasmid, then the coding for the lacZ gene gets disrupted and it cannot produce ?- galactosidase and finally X-gal, which is induced into the medium, will not get converted into a blue product. As a result the colony will be white. If these cells grow in the ampicillin medium the cells containing the insert will produce white colonies and the cells that do not contain the insert will produce blue colonies. Method: 1.95 µl of Frozen competent cells were taken on ice and 5µl of ligation mixture was added to the tube A. 2. The mixture is mixed gently by flicking and the mixture was incubated in the ice for 20 minutes. 3. The mixture was immediately removed from the ice and kept on the heat block for 90 seconds. 4. 400µl of SOC was added to the cells and mixed gently and kept in a large tube for aeration. 5. The mixture was again incubated at 37°C for 1 hour in a shaking incubator. 6. After incubation, 100µl of the cell suspension was transferred to one LB plate containing 100µg/ ml of ampicillin, 0.5mM IPTG and 40µg/ml X-gal. The plates were inverted and incubated at 37°C for 18- 24 hours. A1: heat shock opens the pores present in the cell membrane of E.coli. This enables the plasmid to get inserted into E.coli. after insertion, the cell requires some time for closing the pores that were open and to enable the plasmid to get adopted to e.coli. A2: When E.coli is subjected to a sudden increase of temperature from 0°C to 42°C, the heat shock proteins are formed and they increase the membrane fluidity to enable the plasmids to get inserted into the E.coli. (Wink 2006). A3: Calcium chloride method, Rubidium Chloride method, Cohen method, Hanahan method, Electroporation and colony electroporation methods. (ucdavis.edu 2009). A4: the plates were incubated so as to enable the cells to grow as a single colony and to get distinct colonies from each transformed cell. A5: The recombinant plasmid contains ampicillin resistance gene. So if the recombinant plasmid is present in E.coli, the ampicillin resistant colonies alone will be formed. This will reduce the chance of contamination and easy screening of the cells. Practical 3B: Aim: To analyze the clones using Agarose gel electrophoresis and Southern Blotting technique. Introduction: Specific DNA sequences were identified using Southern Blotting technique. The nucleic acids are separated using the polyacrylamide and Agarose gels. After electrophoresis, the gels are soaked in alkaline solution to denature the dsDNA into ssDNA. The pH of the gel is adjusted and suitable absorbent material used for binding the DNA. Specific DNA within the electrophoretic smear is identified using the hybridization sheet and a suitable probe. Method: 1. In the Electrophoresis method, the sample was prepared as follows: A – pBKS II 2µl + 4 µl 6x gel loading buffer + 18 µl dH2O. D - 10 µl ligation mix + 4 µl 6x gel loading buffer + 6 µl dH2O. 2.To the gel 10 µl of DIG- labeled marker is added. 3. 20 µl of the two samples were loaded on the gel and run at 120 V for 1 hour. 4. After electrophoresis, the gel was removed and kept in the tray containing the denaturation solution for 30 minutes in a rocker with gentle shaking. 5. The gel was then neutralized and soaked in the neutralization solution for 30 minutes on a rocker. 6. Two pieces of filter paper were dipped in the 10x SSC and kept at the bottom of the tray as wick and the gel was kept upside down on the top of the filter papers. 7. A piece of Nylon membrane was kept on the gel and another two strips of wet filter paper are kept above it. Suitable weight is placed on the top of the filter papers and allowed to stand as such for one hour. 8. The gel and the membrane were separated and the membrane was rinsed in 6x SSC. 9. The membrane was air-dried and baked at 120°C in an oven for 30 minutes. 10. 3 µl Pc-ftsZ PCR product us added to the 13 µl of dH2O. 11. The DNA was denatured at 100°C using the heating block and placed in ice immediately. 12. To this mixture 4 µl of High Prime labeling mix is added and centrifuged at low speed and incubated at 37°C for 1 hour. 13. The reaction was stopped by adding 2 µl of 0.2M EDTA. 14. The Hybridization step was carried out and the results were stored. A6: EDTA is used to remove the unwanted radioactive nucleotides from the mix. EDTA being the chelating agent removes the metals from the solution and stops the reaction. A7: Denhardt’s solution can be used as an alternative for EDTA. A8: real time quantitative PCR is an alternate method for Southern Blotting. A9: the advantages of this method are the high through put screening capacity, absence of post-PCR manipulations, large range of accurate quantification in a few hours. (Hoebeeck, Speleman, and Vandesomplele 2007). Practical 4A: Aim: To produce a southern blot and check for the presence of Pc- ftsZ in the vector. Introduction: DNA molecules bind to the probe at different concentrations and the level of stringency to the probe varies due to the variation in the sequence and the absence of exact complementary strand. The membrane is washed with suitable temperature and detergent concentration and also by decreasing the salt concentration. The DIG labeled probe was detected using the anti-DIG antibody conjugated to the enzyme alkaline phosphatase. Method: 1.30 ml of high and low stringency wash was used for the removal of the probe. After this, the membrane was removed from the hybridization bottle and kept on a plastic container. 2. 30 ml of washing buffer was added to the membrane and incubated at room temperature with agitation for 5 minutes. 3. After the removal of washing buffer, blocking buffer was added and incubated as above. The blocking buffer was removed and the 20ml Antibody solution was added at room temperature and agitated. This was followed by 30 ml washing buffer and 20 ml Detection buffer at room temperature. 4. Finally 30 ml of color substrate solution was added to the membrane and placed in a dark room for 16 hours and the results were observed. A1: Vacuum blotting and electro blotting are the alternative method for capillary action in transferring DNA. A2: Pre - hybridization step is required before hybridization step to block the non-specific sites. A3: The probes contain the details of the specific sequence of the DNA which we must detect. Hence the probing of the membrane is required. A4: low stringency washes removes the probes that are bound non-specifically to the membrane. Practical 4B: Aim: To screen the transformed bacteria using pBKS II Pc-fts Z construct. Introduction: Primers T3 and T7 were used for the PCR study as they are complementary to the vector sequences. The colonies were duplicated on a fresh LB agar medium and the PCR is carried out for the single colony of our interest. Method: 1.LB agar plates were taken and marked as 1,2,3,4,5,6,7. 2. The PCR master mix was prepared and kept at cold condition. 3. 20µl of master mix was added to separate 0.2 ml PCR tubes and labeled as 1,2,3,4,5,6,7. 4.A sterile tip was used for the isolation of the white colony and the tip was transferred to the sterile PCR mix. The above procedure was repeated for 2-5 tubes and experiment was started. Similarly a control plate was also prepared. 5. 35 cycles were run in PCR and the freshly prepared LB plates were inoculated and kept at 37°c for 18 – 24 hours. A1: In the denaturation step, the Double stranded DNA is converted into ssDNA and the annealing of the primer occurs during the annealing step and in the elongation step, the dNTPs are bound to the template DNA. A2: In the blue colony, our gene of interest in not present, hence they are used as a control. A3: 2971 bp and 200 bp products are the expected size of PCR products. A4: DNA hybridization, Colony and plaque hybridization, immunological assay and functional complementation are the methods that can be used for gene identification. Practical 5: Aim: To confirm the results of Southern blotting and colony PCR, by running the colony PCR and examine the LB/Amp plates. Method: 1. 6X gel loading buffers were added to the PCR tubes and the samples were loaded onto the 1.5 % Agarose gel and run at 1202 V for 45 minutes. 2. The LB/Amp plates and Southern Blots were examined and the results were noted. The molecular weight of the given bands was compared with the DIG-labeled marker. 3. After the PCR reactions are complete, the gel results were observed under the UV light. Result : The above figure shows the Agarose gel electrophoresis image for the southern blot performed with DIG markers. The wells were loaded with pBKS, Linear pBKS, insert and ligated sample in lanes 2, 3, 4, ands respectively. The other lanes show the other batch results. Discussion: Southern blot is used to verify whether the DNA fragment or gene of interest is present in the sequence or not. When the sample was digested with the same restriction enzymes BamHI and Hind III, the same pattern of bands were seen. The DNA fragment of our interest was not properly well into the clone and this is confirmed by the thick linear pBKS II DNA having a higher molecular weight. A single DNA band is observed in the gel which indicates that the DNA appears as a smear. The restriction enzyme was not able to cut the DNA into fragments indicating some error in the experiment. Answer 1: Immunological assay can be used for the detecting the specific DNA sequence on a southern blot. Answer 2: 3 bands. Answer 3: After transformation, transduction can be carried out. References: Boyer, RF., 2000. Modern Experimental Biochemistry, Pearson Educational India. Hoebeeck, J., Speleman, F., and Vandesomplele, J., 2007. Real-time quantitative PCR as an alternative to Southern blot or fluorescence in situ hybridization for detection of gene copy number changes, Methods in Molecular Biology, Vol.353, pp.205 – 226. Robert, RJ and Murray, K., 1976. Restriction Endonucleases, Critical reviews in Biochemistry and Molecular biology, Vol.4, no.2, pp.123- 164. Siwach, P and Singh, N., 2007. Molecular Biology, Firewall Media. ucdavis.edu., 2009. Protocols. [Online] Available at http://rothlab.ucdavis.edu/protocols/protocols.html (Accessed on May 28, 2012) Westermeier, R., 2006. Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations, John Wiley and Sons. Wink, M., 2006. An introduction to Molecular Biotechnology: Molecular fundamentals, methods and applications in modern Biotechnology, Wiley- VCH. , Read More