Plant Biotechnology
LABORATORY EXERCISE Agarose Gel Electrophoresis Introduction Agarose gel electrophoresis is a process that undertakes biochemistry and molecular biology understandings to identify and analyse DNA and RNA strands. This is done by separating the genetic material by its size. The genetic material is placed in the solidified wells of the agarose (a linear polymer composed of alternating isomers of the sugar galactose) at the cathode end. The negatively charged nucleic acid molecules move through the agarose matrix with the assistance of an electric field (electrophoresis). This is because genetic material is negatively charged, and will move towards the anode when current is passed through. The shorter molecules migrate faster than the longer molecules. The use of electrophoresis buffer in the making of the agarose gel is to establish a constant pH and to provide ions to support the conductivity. If instead water was used, then the genetic material will not migrate during the electrophoresis. The amount of voltage used is crucial to the migration of the genetic material. When increasing voltage is applied to the gel, larger fragments migrate proportionally to that of the smaller fragments. Thus, the voltage applied is usually 5 volts per centimetre to the gel. The gel is then immersed in ethidium bromide, a fluorescent dye that covalently binds (intercalates) between the bases of nucleic acid. Then UV light is passed through the gel to make the genetic material visible. Materials TAE Buffer or TBE Buffer (1x); 6x loading dye (Fermentas/Promega); agarose powder; standard DNA ladder marker; sterile ddH2O; 10mg/ml EtBr stock solution. Methodology 1. 0.5g of agarose powder was added with 50ml of 1x TBE in a small Erlenmeyer flask. This gives 1% gel (The higher the % of gel, the better the separation of DNA bands). 2. The flask was microwaved until agarose dissolved completely. Solution was checked occasionally to avoid boiling from occurring. 3. The flask was cooled to 50°C by running it under cold tap water. A comb was then placed after pouring the agarose. 4. The gel was left to solidify, this process can be fastened by placing the gel rig in a refrigerator 5. After the gel is solidified, the comb was gently removed by pulling it evenly upward 6. Gel mould was positioned inside the electrophoresis chamber and covered with running buffer (1x TBE). 7. DNA samples are mixed with loading dye on a parafilm. (Samples and dyes need to be diluted accordingly; e.g. 5μl sample + 1μl 6x loading dye or 2μl sample + 3μl ddH2O + 1μl 6x loading dye).
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Plant Biotechnology
8. With the use of a micropipette, the sample mixture and DNA ladder was carefully loaded into respective wells. Poking the bottom of the gel was avoided. 9. A cover was placed on the electrode box and the gel was electrophoresed at 120V for 45 minutes 10. Once the loading dye reached approximately ¾ of the gel, the power pack was switched off. 11. Gel was carefully removed from mould and placed it into the staining box containing EtBr for a few minutes. Gloves were worn because EtBr is mutagenic. 12. DNA stained with EtBr was placed on a transilluminator. The UV light was then switched on and the DNA bands were visualized. 13. After visualizing of DNA was completed, the gel was disposed in a proper biohazard waste bucket and the surface of the transilluminator was cleaned with distilled water. Results
Done in Biochemistry Laboratory: from right, Lane 2 Muricata paniculata and Lane 3 Canna glauca. Lane 2 showed a fluorescent clear band and the smear at the background shows there is DNA denaturation; Lane 3 has vast RNA and protein contamination.
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Plant Biotechnology
Done in Plant Tissue Culture Laboratory: far left, Lane 11 Zinnia elegans showed clear band of DNA with little denaturation and some protein and RNA contamination. Discussions DNA bands and its quality The DNA denaturation which causes smear occurs because, whenever a whole plant genomic is cleaved with an enzyme, usually a smear of DNA fragments can be seen on agarose gel after electrophoresis. And normally, clear, discrete bands appears on the background of smear. In many plant genomes, 20-25% of cytosine are methylated. As a result, when a restriction enzyme is used to cleave the DNA fragments, various sizes of fragments are formed and the methylated cytosine produces larger DNA fragments (Hess et al., 1988). Therefore, it is noticeable that when electrophoresis are being conducted, smaller DNA fragments travels at a faster rate and larger DNA fragments travel at lower rate. RNA contamination occurs because RNA has similar structure as the DNA as RNA is nucleic acid as well. Since RNA is of smaller molecular weight, the RNA fragments travels at a faster rate compared to DNA. A standard RNase digestion could be done to eliminate the RNA (Kieleczawa, 2006). As for protein contamination, we can say that one of the leading factor would be insufficient phenol extraction. Solvents like phenol or chloroform helps to denature proteins. Usage of isoamylalcohol helps to separate precipitated proteins and the nucleic acids remains in the aqueous phase. Kang et al. (1998) reported grinding and lysis of dry seed and incubation with buffer containing proteinase K gave DNA of good quality and quantity. Usage of proteinase K helps to overcome shearing of DNA and loss of yield due to centrifugation by digesting polypeptide into smaller molecule which can be easily removed by phenol extraction. 3
Plant Biotechnology
Other ways to improve visualization quality is by increasing concentration of DNA sample and using SYBR Green I or SYBR Safe instead of ethidium bromide – 25 times more sensitive and very less mutagenic. Why no bands? 1. There is a chance where the buffer or agarose used are expired and lose its activeness. 2. Pellet which supposed to contain the DNA samples are thrown out in previous experiment. 3. Correct technique during isolation of DNA experiment was not followed. 4. Amount of voltage applied is too low. 5. There is a possibility the percentage of agarose used are too low, thus unable to produce and visualize DNA samples. TAE and TBE buffer Tris-acetate-EDTA (TAE) buffer is a most common buffer solution that consist a mixture of Tris base, acetic acid, and EDTA. It is used for agarose electrophoresis analyses of DNA products resulting from PCR amplification, DNA purification protocols, or DNA cloning experiments, with and without sodium chloride. Tris-Borate-EDTA (TBE) buffer solution containing a mixture of Tris base, boric acid and EDTA. It is also often used for agarose electrophoresis analyses of nucleic acids products. TAE is best for linear, double stranded, or large pieces of DNA which more than 20kb but it requires to be replaced frequently for those more than 4 hours gel run times. TBE is much preferable for separation of smaller DNA fragments such as restriction enzyme digest. In the other hand, TBE has greater ionic strength and buffering capacity, allowing us to have sharper resolution compared to TAE, but it is also more costly and inhibits DNA ligase which causes problems if subsequent DNA purification and ligation steps are intended as limitation. The use of sodium chloride in TAE may retards DNA mobility and lead to incorrect interpretations of resulting DNA banding pattern. Functions of the chemicals Agarose (agarose gel) is a polymeric cross-linked polysaccharide extracted from the seaweed agar. The agarose forms a porous lattice in the buffer solution and the DNA must slip through the holes in the lattice in order to move toward the positive pole. This slows the molecule down. Larger molecules will be slowed down more than smaller molecules, since the smaller molecules can fit through the holes easier. As a result, a mixture of large and small fragments of DNA that has been run through an agarose gel will be separated by size. Agarose was used widely in gel electrophoresis because it gels at a lower temperature, does not contain the inhibitors of virus growth frequently present in agar, and has more uniform pore size than that of agar. It also easily poured, does not denature the samples. The samples can also be recovered. The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and
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Plant Biotechnology
different forms of genetic material may run in unpredictable forms. Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. Most agarose gels are made with between 0.7% (good separation or resolution of large 5-10kb DNA fragments) and 2% (good resolution for small 0.2-1kb fragments) agarose dissolved in electrophoresis buffer. 1% gels are common for many applications. Loading dye plays essential role in obtaining sharp DNA bands. It serves three vital functions: it is used to terminate enzymatic reactions before electrophoresis (stop solution - usually provided by EDTA), provide density for loading the sample into the well (provided by glycerol or sucrose), and it provides the way monitoring the progress of electrophoresis. EDTA, however it is not sufficient for fully dissociating DNA-protein complex. Urea, at a concentration of 5M, is the best protein-denaturing agent because it does not interact with agarose and affecting DNA mobility. Addition of Ficoll-400 may help increase clarity of bands produced. Marker is a set of DNA fragments of known molecular sizes that are used as a standard to determine the sizes of unknown fragments. In addition it can be used to approximate the mass of a band by comparison to a special mass ladder. The 1kb ladder with fragment ranging from about 0.5kbp to 10 or 12kbp and the 100bp ladder with fragments ranging from 100bp to just above 1000bp are the most frequent. DNA ladders are often produced by a suitable restriction digest of a plasmid. There are special DNA ladders for supercoiled DNA and RNA. Ethidium bromide or EtBr is the most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis. It fluoresces under UV light when intercalated into DNA (or RNA), of which it decreases DNA mobility up to 15 percent. By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than 20ng DNA becomes distinctly visible. EtBr is a known mutagen; however, safer alternatives are available such as SYBR Green I and SYBR Safe (it is more expensive, but up to 25 times more sensitive). Tips 1. Check expiry date of agarose gel and other chemicals before using it to avoid errors. 2. Avoid creating bubbles when pouring agarose gel into the comb. 3. Pellet to be used must be sterile to avoid contamination and error. 4. Be gentle and caution when inserting the samples into the well and avoid poking the agarose gel because it may cause leakage. 5. Put on glove as protection before staining process because EtBr is very carcinogenic. Conclusion From this experiment, we become aware and understood on how agarose gel electrophoresis works.
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Plant Biotechnology
References Barker, K. 2005. At The Bench: A Laboratory Navigator. Cold Spring Harbour Laboratory Press, New York, USA. Chawla, H.S. 2002. Introduction to Plant Biotechnology. Science Publisher, Enfield, New Hampshire, USA. Hess, W.M., Singh, R.S., Singh, U.S., and Weber, D.J. 1988. Experimental and Conceptual Plant Pathology. Gordon and Breach, New York, USA. Henry, R.J. 2008. Plant Genotyping II: SNP Technology. CABI International, Cambridge, Maasachusetts, USA. Hames, B.D. and Hinggins, S.J. 1995. Gene Probes: A Practical Approach. Oxford University Press, New York, USA. Kang, H.W., Cho, Y.G., Yoon, U.H., and Eun, M.Y. 1998. A Rapid DNA Extraction Method for RFLP and PCR Analysis From A Single Dry Seed. Plant Molecular Biology Reporter. 16 (2): 90-94. Karp, A., Isaac, P.G., and Ingram, D.S. 1998. Molecular Tools for Screening Biodiversity: Plants and Animals. Kluwer Academic Publisher, Dordrecht, Netherlands. Kieleczawa, J. 2006. DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett Publishers, Sudbury, Massachusetts, USA. Kumar, A and Garg, N. 2005. Genetic Engineering. Nova Science Publisher, New York, USA. Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual Volume 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. Surzycki, S. 2003. Human Molecular Biology Laboratory Manual. Blackwell Science, London, UK. http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html (300909) http://comp.uark.edu/~mlehmann/Agarose.pdf (300909) http://www.molecularstation.com/dna/dna-gel-electrophoresis/ (300909) http://www.plantpath.cornell.edu/glossary/Defs_A.htm (031009) http://en.wikipedia.org/wiki/TAE_buffer (300909)
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Plant Biotechnology
http://en.wikipedia.org/wiki/TBE_buffer (300909) http://biotech.about.com/od/buffersandmedia/ht/TAE.htm (011009) http://biotech.about.com/od/buffersandmedia/ht/MakeTBE.htm (011009)
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