DOI: http://dx.doi.org/10.18203/2320-6012.ijrms20160012

Optimized genomic DNA extraction by a modified organic phenol- chloroform method without using PCR for best results

Hamid Ali, Sadaf Jafar, Qurat-ul Ain

Abstract


Background:  The objective of the study was to design a cost effective, efficient and better protocol for genomic deoxyribonucleic acid (DNA) extraction.

Methods: This was an experimental study, which is carried out ARID Research laboratory. The duration of study was four months from March 2015 to June 2015. White blood cells were used for extraction of DNA. Two procedures RBC lysis and extraction of DNA from the pellet was done. The goal was to obtain high quality and quantity DNA yield. Even the amount of blood less than 1 ml can be used for extraction. The extraction of DNA comprise of disruption of nuclear membrane and removal of protein from the pellet. After extraction DNA was purified. DNA was confirmed with the help of Nanodrop technology. Genomic DNA was also confirmed by using horizontal agrose gel electrophoresis and visualized using Gel Doc System. Whole blood approximately 10ml µg, yields 250 µg of DNA, using latest organic phenol chloroform method.

Results: Successful extraction of DNA from the WBCs without compromising quality and quantity was the end result. Shelf life of DNA was increased. Current modified protocol of DNA isolation, extraction and purification of DNA is not only cost-effective and highly yielding but also reproducible.

Conclusions: The DNA extracted with modified organic phenol- chloroform method was of high quality and quantity. The bands of DNA were confirmed with horizontal electrophoresis. Clear DNA band were visualized indicating the precision of latest protocol. Another important advantage was that very small amount of blood sample is required for DNA extraction and the protocol was cost effective and efficient. Present protocol will be very beneficial for genomic studies, requiring DNA extraction. It will enable researchers to work with fewer budgets and less sample size, obtaining best results.


Keywords


DNA, Extraction, WBCs

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