Discriminatory Power of Agarose Gel Electrophoresis in DNA Fragments Analysis

Lee, Seow Ven; Bahaman, Abdul Rani

doi:10.5772/36891

Cited by 7 publications

(5 citation statements)

References 23 publications

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“…For optimal resolution, horizontal gels should ideally be cast no thicker than 3-4 mm. Thicker gels, such as those with a thickness of 10 mm, may result in reduced clarity and resolution, leading to a haziness in the visualization of smaller DNA fragments when compared to a 3 mm thick gel, which provides more consistent resolution throughout the gel [12].…”

Section: Resultsmentioning

confidence: 99%

“…With a larger comb, more volume may be inserted into the well, but the DNA bands may be broader. A comb with a width of 1 mm will produce more distinct DNA bands [12], [13]. The amount of DNA that can be loaded onto a gel is influenced by two main factors: the distribution of DNA fragments (including the number and size of target fragments) and the capacity of fragments are larger in size (>10 kb), there is a risk of overloading the gel, which can result in undesirable effects such as lagging and smearing of the DNA bands.…”

Section: Resultsmentioning

confidence: 99%

“…The amount of DNA that can be loaded onto a gel is influenced by two main factors: the distribution of DNA fragments (including the number and size of target fragments) and the capacity of fragments are larger in size (>10 kb), there is a risk of overloading the gel, which can result in undesirable effects such as lagging and smearing of the DNA bands. This is because larger DNA fragments require more space to migrate properly through the gel matrix, and when the capacity of the well is exceeded, the DNA can have trouble in moving at the desired pace and may exhibit irregular migration patterns [12]. Therefore, it is important to consider the size of the DNA fragments and the well capacity to prevent overloading issues and ensure accurate and clear separation of the DNA bands during gel electrophoresis.…”

Section: Resultsmentioning

confidence: 99%

“…It's important to note that freezing the samples does not halt the action of DNAase, leading to a reduction in DNA concentration. When frozen samples were held at -20 °C for a duration of 10 days, approximately 65% loss of DNA concentration and a 90% decrease in the amount of extracted DNA were observed [12,14].…”

Section: Resultsmentioning

confidence: 99%

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Methylation Specific PCR (MSP): Nested PCR vs Unnested PCR

Humardani,

Mulyanata,

Tanaya

et al. 2023

Advances in Biological Sciences Research

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Methylation-specific PCR (MSP) is a valuable technique for studying DNA methylation patterns due to its straightforward design and implementation, high sensitivity in detecting methylated DNA, and ability to analyze large sample sizes cost-effectively rapidly. However, researchers need to be cautious when working with new samples or samples stored in the freezer for an extended period. Freezing does not prevent the action of DNAase enzymes, which can lead to reduced DNA extraction yields. During MSP, DNA undergoes changes, and PCR is performed using two sets of primers that specifically target methylated and unmethylated DNA regions. The use of bisulfite conversion treatment, an essential step in methylation analysis, presents significant challenges. One challenge is that bisulfite treatment can cause DNA fragmentation, particularly when the initial DNA concentration is low. In nested MSP, where an additional round of PCR is performed, smearing may occur due to the high DNA concentrations used. Therefore, for new samples with a DNA concentration of 1-10 µg/µL, it is recommended to use the unnested MSP technique prior to bisulfite conversion treatment.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Methylation Specific PCR (MSP): Nested PCR vs Unnested PCR

Humardani,

Mulyanata,

Tanaya

et al. 2023

Advances in Biological Sciences Research

View full text Add to dashboard Cite

show abstract

“…For this purpose, 0.7% agarose gel (w/v) was prepared (Ghatak et al, 2013;Fuentes-Pardo & Ruzzante, 2017). For a better gel result, a thin and narrow gel comb was preferred (Lee & Bahaman, 2012;Arslan et al, 2021). 10 μl isolated DNA samples were loaded into wells and run at 55 volts for 240 min.…”

Section: Agarose Gel Electrophoresis Of Isolated Dna Samplesmentioning

confidence: 99%

Donmuş Çözünmüş Kanın Farklı Değerlerde Santrifüjünün DNA İzolasyonu Üzerine Etkileri

Arslan

2023

Yüzüncü Yıl Üniversitesi Fen Bilimleri Enstitüsü Dergisi

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DNA isolation from blood is a commonly used application to obtain nDNA and mtDNA. It was previously shown that DNA isolation could be performed from the pellet obtained after centrifugation of freeze-thawed blood (FTB), and this pretreatment had constructive results on DNA isolation. However, which centrifugation levels can be used for this pretreatment and their effects are unknown. The aim of the study was to determine appropriate centrifugation levels for this pretreatment and show their effects on isolated DNA. For this purpose DNA isolations were carried out from both pellet and supernatant obtained by centrifugation at different levels of FTB. Then, spectrophotometric, gel electrophoresis, and real-time PCR analyses were performed in the isolated DNA samples. As a result, centrifugation of FTB at 5 000 ×g for 2 min or over let genetic material to pellet completely, which enable to obtain higher DNA yield. Real-time PCR results showed that mtDNA/nDNA ratios did not change in the isolated DNA samples which were performed from pellets obtained by defined centrifugation levels whereas isolated DNA integrity decreased. To conclude, centrifugation at 5 000 ×g for 2 min or over can be used to harvest and wash genetic material found in FTB before DNA isolations.

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Yield Gel via Quantitative Gel Electrophoresis

Parks

Torres

2023

Methods in Molecular Biology

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Discriminatory Power of Agarose Gel Electrophoresis in DNA Fragments Analysis

Cited by 7 publications

References 23 publications

Methylation Specific PCR (MSP): Nested PCR vs Unnested PCR

Methylation Specific PCR (MSP): Nested PCR vs Unnested PCR

Donmuş Çözünmüş Kanın Farklı Değerlerde Santrifüjünün DNA İzolasyonu Üzerine Etkileri

Yield Gel via Quantitative Gel Electrophoresis

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