Extraction of DNA from Agarose Gels

Downey, Nicholas

doi:10.1385/1-59259-409-3:137

Cited by 6 publications

(6 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Examples include physical methods such as electrophoresis into a preformed trough, enzymatic methods such as agarose digestion, or purification methods using columns or magnetic beads. 5,18 Although in theory it is possible, given the specificity of the sequencing primers, to generate NA sequence information from a single amplicon included in a multiplex PCR reaction, this is an approach that is prone to primer interference, lower product yields, and increased analysis complexity, and therefore is not recommended for diagnostic applications. In most cases, repeating the PCR reaction using one specific pair of primers will resolve the problem.…”

Section: Preparing Dna Template For Sequence Analysis Submissionmentioning

confidence: 99%

“…Numerous options are available for this PCR product purification step, including bead-based, column-based, and enzymatic methods, all of which are available commercially. 5,18 For VDLs that outsource their sequencing, commercial sequencing laboratories typically provide, via their websites, a list of recommendations or preferences for specific clean-up kits and procedures to be used prior to submitting samples to their facility for sequencing. Many sequencing facilities also provide PCR purification services for an additional fee.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Guidelines for Sanger sequencing and molecular assay monitoring

et al. 2020

View full text Add to dashboard Cite

Genetic sequencing, or DNA sequencing, using the Sanger technique has become widely used in the veterinary diagnostic community. This technology plays a role in verification of PCR results and is used to provide the genetic sequence data needed for phylogenetic analysis, epidemiologic studies, and forensic investigations. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians has prepared guidelines for sample preparation, submission to sequencing facilities or instrumentation, quality assessment of nucleic acid sequence data performed, and for generating basic sequencing data and phylogenetic analysis for diagnostic applications. This guidance is aimed at assisting laboratories in providing consistent, high-quality, and reliable sequence data when using Sanger-based genetic sequencing as a component of their laboratory services.

show abstract

Section: Preparing Dna Template For Sequence Analysis Submissionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Guidelines for Sanger sequencing and molecular assay monitoring

et al. 2020

View full text Add to dashboard Cite

show abstract

“…The PCR product was run on a 1% agarose gel and visualized under UV light. The corresponding band to the amplified sequence was extracted and purified from the gel according to Downey, 2003.…”

Section: -Culture Mediamentioning

confidence: 99%

Genetic Transformation of Bacillus licheniformis by Gene Responsible for α-Amylase Production in Media Contains Sugar Crops Wastes

Khaled

El-Aziz

Badr³

2018

Journal of Agricultural Chemistry and Biotechnology

View full text Add to dashboard Cite

The importance of amylase enzymes is attributed to their ability to catalyze hydrolysis of starch and polysaccharides into maltose, glucose and/or maltodextrin for industrial processes. Their thermophilic characteristics are necessary to maintain stability under different conditions. Amylases are produced by, fungi, higher plants and animals. In the present study, thermophilic Bacillus (Bacillus stearothermophilus) isolated from beet pulp during sugar processing was used for cloning α-amylase gene into Bacillus licheniformis. The cloned and sequenced gene was 1827 bp in length. It has been registered in the GenBank (accession no. LC259133.1). This thermophilic amylase, with a molecular weight of 60 kD, was expressed and purified from the recombinant strain and matched to the α-amylase family protein of Bacillus stearothermophilus (GenBank accession no. M57457.1) using the NCBI database. The amino acid sequence of the cloned recombinant enzyme was 98% similar to that of the Bacillus stearothermophilus enzyme in the database. Bacillus strains were cultured and the activity of the enzyme was determined. The purified amylase on sugarcane bagasse and/or sugarbeet pulp media was highly active. Moreover, the modified B. licheniformis surpassed the other strain in cell mass amount and amylase activity on all media. These results confirmed that the gene transferred into B. licheniformis effectively increased amylase activity in this bacterium, while sugarcane wastes increased cell mass amount and enzyme activity. Also, thermostability of α-amylase was not clearly different between modified and original B. licheniformis. These provided evidence that gene transfer to B. licheniformis effectively increased its amylase activity but did not affect its thermostability. The enzyme produced in this study have high thermostability even 90 °C, high thermostability range allows it to be utilized in industrial applications, because their high yield, as well as their time and cost saving.

show abstract

“…PCR mixture prepared and the reaction performed, then the product was run on 1 % agarose gel and visualized under the UV light. Agarose gel piece including the amplified gene region was extracted from gel and purified as described by Downey (2003). The amplified PCR product was digested using restriction enzymes and ligated into pET15b vector using the TA cloning kit vector (Invitrogen, USA).…”

Section: Dna Extraction Plasmids Construction and Cloning Of Amylasementioning

confidence: 99%

Evaluation of Amylase Activity Produced by Genetically Modified Bacillus Grown on Different Media Containing Sugar Crops Wastes

Khaled

Kash

El-Aziz

et al. 2017

Journal of Agricultural Chemistry and Biotechnology

View full text Add to dashboard Cite

In this investigation, α-amylase gene region from a thermophilic Bacillus stearothermophilus isolated from sugar beet juice obtained from Dakahlia Sugar factory (Egypt) was cloned to competent Bacillus licheniformis ATCC 27811 cells. The original and modified Bacillus strains were spread on different media composition and enzyme activity was determined. According to the results, the α-amylase enzyme was seen to have highest activity at media containing sugarcane bagasse and/or sugar beet pulp. The results revealed that modified Bacillus surpassed the original one at all studied media. This obtained result proofed that gene transferred into BL had effective action on increase α-amylase activity. The thermo-stability studies revealed that enzyme activity of modified bacillus was high for the first 8 hours at 90°C, it was ranged from 95-90 % after 6 hours (enzyme activity decreased from 7.80 to 7.02 gm glucose/ml) then, decreased to 65-63 % at the end of 24 hours.

show abstract

Extraction of DNA from Agarose Gels

Cited by 6 publications

References 0 publications

Guidelines for Sanger sequencing and molecular assay monitoring

Guidelines for Sanger sequencing and molecular assay monitoring

Genetic Transformation of Bacillus licheniformis by Gene Responsible for α-Amylase Production in Media Contains Sugar Crops Wastes

Evaluation of Amylase Activity Produced by Genetically Modified Bacillus Grown on Different Media Containing Sugar Crops Wastes

Contact Info

Product

Resources

About