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Electrophoresis

Autor:   •  March 14, 2018  •  1,318 Words (6 Pages)  •  99 Views

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It was recorded ‘was not visible’ for some bands but it should be noted that the bands which counted as visible appeared like large smears and some were not properly visible either. Hence, It could be assumed that more than one band was present in these large smears. Band 1, 2, 3 and 4 are not very distinct from each other and therefore it can be estimated roughly based on the size of the smear that there were at least 2 bands in one smear. DNA degradation by nucleases, improper electrophoresis conditions and gel shift effect are reasons of obtaining smeared DNA bands. (Smith, 1996). This also accounts for the difference in the calculated value for the fragments and their ideal values.

If we look at the migration pattern of the DNA fragment, we can say that it is atypical. DNA fragments migrate differently due to differences in DNA sequences. AT rich DNA may migrate slower than an equivalent size GC rich DNA fragment. (Hirano, 2010). The sequences of Fermentas DNA ladders are chosen to allow for highly accurate DNA migration according to size; however, due to differences in nucleotide sequence or the overall DNA structure, sample migration can sometimes slightly differ from ladder band migration. Another reason for such atypical migration pattern is due to improper electrophoresis conditions where excessive electrophoresis run times or voltage may result in migration of small DNA fragments off of the gel. (Agarwal & Singh, 2010). Very short or slow electrophoresis may result in incompletely resolved bands.

Restriction endonucleases (Restriction enzymes) are synthesized by many bacterial species to bind and cleave foreign DNA. They recognize precise DNA sequences and cut both the strands of DNA at the binding site resulting in large fragments that get rapidly degraded by exonucleases before its gene expression is expressed. The restriction enzyme used in this experiment, HindIII is known for catalyzing staggered cleavages that result in DNA fragments with single-stranded extensions (Sticky ends) and as they are complementary, they comprise the ability to re-form a double-stranded structure. HindIII is produced by Haemophilus influenzae Rd and its recognition sequence is A*↓AGCTT. HindIII cuts the λ DNA sequence into eight fragments between the two Adenine bases of the DNA sequence .Restriction enzymes are useful in manipulating DNA and developing restriction maps of DNA which is useful for identifying fragments of DNA that contain specific genes. (Hirano, 2010). They also play an important role in recombinant DNA technology. Mapping of restriction sites allowed identification of sites of mutations (variations) in the genome of a population which allows identification of an individual in a large heterogeneous population. This experiment used #SM0102 LambdaDNA/ HindIII restriction enzyme produced by the Fermentas Company.

GelRed is a sensitive, stable and environmentally safe fluorescent nucleic acid dye designed to replace the highly toxic ethidium bromide (EB) for staining dsDNA, ssDNA or RNA in agarose gels or polyacrylamide gels. GelRed is far more sensitive than EB without requiring a destaining step. As nucleic acid binding dyes can affect DNA migration during electrophoresis, post-staining of gels is highly recommended. Thus, post-staining with GelRed results in superior sensitivity and eliminates the possibility of dye interference with DNA migration.

In general, as recommendations, we must let the gel to run for about 10 more minutes, allowing the bands to separate more in order to form more distinct bands for especially those bands which are very heavy. (Hirano, 2010). In addition, fresh electrophoresis buffers, freshly poured gels must be used to minimize nuclease contamination of DNA solutions to get rid of smeared bands. Another recommended solution that must be used to overcome the problem of smeared DNA bands is that, when inserting the comb into the gel, we have to make sure that it is vertical to the gel surface and stable during gel casting and its solidification. (Smith, 1996).

They are several application of agarose gel electrophoresis. For example, it can be used to estimate the size of DNA molecules following restriction enzyme digestion. Also, it is used in analyzing of PCR products such as in molecular genetic diagnosis or genetic fingerprinting.( Smith, 1996). Chemists also use it to separate restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer. Agarose gel electrophoresis together with restriction digestion is most preferred technique to identify positive clones of cloning experiments. In addition, garose gel electrophoresis is often used to purify specific fragments. To purify a specific fragment, samples are run on agarose gel. Fragments of interest are excised and eluted by various techniques. Since agarose gel electrophoresis fractionate the DNA fragments on the basis of size, similar size DNA fragments cannot be purified by agarose gel electrophoresis.

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References

Agarwal S & Singh S (2010) Study of metal complexes of glycil glycine by paper electrophoresis. Oriental Journal of Chemistry. Vol. 26, page:1223-1225

Hirano K (2010) Analysis of supercoiled DNA by agarose gel electrophoresis using low-conducting sodium threonine medium. Analytic Biochemistry. Vol. 400, page: 148-150

Smith D (1996) Agarose Gel Electrophoresis. Molecular Biology, Vol. 58, page. 17-21

Thermo Fisher Scientific. (2013a). GeneRuler 1 kb Plus DNA Ladder, Ready-to-Use 75 to 20,000 bp. Available: http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-plus-dna-ladder-ready-to-use-75-to-20000-bp/. Last accessed 2nd April 2013.

Thermo Fisher Scientific. (2013b). Lambda DNA/HindIII Marker, Ready-to-Use. Available: http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/lambda-dna-hindiii-marker-ready-to-use/. Last accessed 7th April 2013

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