Development of a chamber system for rapid, high yield and cost-effective purification of deoxyribonucleic acid fragments from agarose gel

Abstract

Background: There are several methods commonly practicing for deoxyribonucleic acid (DNA) purification from agarose gel. In most laboratories, especially in developing countries, present methods for recovering of DNA fragments from the gel are mostly involved organic solvents. However, manual purification using organic solvents are toxic, labor intensive, time consuming and prone to contamination owing to several handling steps. The above mentioned burdens as well as cost and long time to import them, especially in developing countries, prompted us to design and develop a chamber system for rapid, non-toxic, cost-effective and user friendly device for polymerase chain reaction (PCR) products purification from agarose gel.

Materials and methods: The device was made from plexiglass plates. After amplification of two fragments of 250 and 850 bp, PCR products were electrophoresed. Subsequently, the desired bands were excised and purified with three method: HiPer Mini chamber, phenol extraction method and spin column procedure. To assess the suitability of the purified DNAs, restriction digestion was applied.

Results: Results showed that the yield of recovered DNA in our method was above 95%, whereas the yields obtained with conventional phenol extraction and spin column methods were around 60%.

Conclusion: In conclusion, the current method for DNA elution is quick, inexpensive and robust and it does not require the use of toxic organic solvents. In addition, the purified DNA was well has suited for further manipulations such as restriction digestion, ligation, cloning, sequencing and hybridization.

Keywords: Gel electrophoresis; phenol extraction; polymerase chain reaction product purification.

Figure 1

Schematic representation of the device in various orientations. I: The device which was made from plexiglass; II: Various parts of the device

Figure 2

Agarose gel electrophoresis of the purified PCR products of 850 bp with three methods. Lanes 1-2: Spin column method; lanes 3-4 and 7-10: Conventional phenol extraction; lanes 5-6: Current optimized procedure with our device; lane 11: 100 bp DNA ladder

Figure 3

Restriction enzyme (AvaII) digestion of DNA fragments purified using three different methods. Lane1: ladder 50 bp; Lane 2: conventional phenol extraftion; Lane 3: Spin column method; Lane 4: current optimized procedure with device

Figure 4

Plasmid purification with three methods. From the left side, Lanes 1-2: Spin column method; Lane 3: Conventional phenol extraction; Lanes 4-5: Current optimized procedure with our device