IGEM:Hong Kong HKUST/Investigations/Effect of agarose concentrations on separation of linear DNA in gel electrophoresis

Authors Astrid S. Alim Nadia Benedicta Bongso Angeline Widjaja Description Investigation of the effect of agarose gel concentration on the separation of different size of linear DNA to find the best gel concentration for separation of certain range of DNA size. Abstract Electrophoresis is a method to separate substances based on the rate of movement under an electric field. Agarose is a polysaccharide purified from seaweed. The DNA is pulled through the pores in the gel by electrical current. DNA will move toward the positive electrode and away from the negative electrode. There are several factors affecting the speed of DNA movement during electrophoresis such as: strength of electrical field, concentration of agarose gel and size of the DNA molecules. Smaller DNA molecules move faster than the larger ones. DNA itself is not visible within the gel and should be visualized using a dye that binds to DNA. Intended Result With constant voltage and running time, separation of longer DNA will be clearer with a lower agarose gel concentration whereas separation of shorter DNA will be clearer with a higher agarose gel concentration. Method Instead of using other DNA, we used commercial DNA ladder. DNA ladder has more different sizes of linear DNA than self-cut extracted linear DNA. Results Figure 1. 0.5% gel concentration Figure 2. 1.0% gel concentration All bands in 1 kb DNA are clearly separated. Bands in 100 bp are still stacked together. Bands in 100 bp are more clearly separated in this gel. Figure 3. 1.5% gel concentration Figure 4. 2.0% gel concentration The bands in tail area of 1 kb DNA become stacked whereas bands in 100 bp are all clearly separated. More bands in tail area of 1 kb DNA become stacked. Bands in 100 bp are fully separated. Figure 5. 2.5% gel concentration Bands in 1 kb DNA are stacked together. Conclusion: Separation of 1 kb DNA is best observed with 0.5% gel concentration. Separation of 100 bp DNA is best observed with 2.0% gel concentration. Discussion The separation of molecules within a gel is determined by the relative size of the pores formed within the gel. As the total amount of agarose powder increases, the pore size decreases. The pore size of the gel decreases due to increased rate of nucleation and closer packing of the chain. Shorter DNA, which is 100 bp, moves faster and migrates farther than longer ones in higher concentration because shorter DNA migrates more easily through the decreasing pore size of the gel. According to the result photo, 100 bp DNA ladder is best observed with 2.0% concentration as the bands are separated clearly, while in other gel concentration, the 100 bp DNA ladder is stacked together. Longer DNA, which is 1 kb, moves faster and migrates farther than shorter ones in lower concentration because longer DNA migrates more easily through the increasing pore size of the gel. According to the result photo, 1 kb DNA ladder is best observed with 0.5% concentration as the bands are separated clearly, while in other gel concentration, the 1 kb DNA ladder stacked together and become smear. This phenomenon is called sieving. Materials 1 kb Generuler DNA ladder standard 100 bp Generuler DNA ladder standard DNA sample loading dye Agarose gel 1X TAE Buffer GelRed 10,000X as DNA stain Electrophoresis machine (chamber & power supply) Gel casting tray and combs Pipette and tips Procedures Pre-Stain Gel Electrophoresis 1. Measure 0.1 gram agarose powder using the electronic balance for 0.5% gel concentration 2. Measure 20 ml of 1X TAE using the measuring cylinder 3. Pour the agarose powder and 1X TAE into a conical flask 4. Mark the top of the solution using the marker 5. Heat the solution in the microwave for 50 seconds 6. Add distilled water to replace the evaporated buffer 7. Cool the solution down in the water bath until it is in a lukewarm condition 8. Add 1 µL of GelRed using the pipette 9. Pour the solution into the gel tray, put the comb, and wait until it becomes an agar / gel 10. Put the agar in the gel tray inside the electrophoresis machine 11. Insert the 6 µL 1 kb DNA ladder (1 µL 1 kb DNA ladder, 1 µL 6X LD , 4 µL H2O) and 10 µL 100 bp DNA ladder (1 µL 1 kb DNA ladder, 1 µL 6X LD , 8 µL H2O) into the gel using the pipette 12. Set the system with 135V and run the gel for 20 minutes 13. Take a photo of the gel using the UV light 14. Do step 2-12 with different weight of agarose powder to make gel with different concentrations 0.2 gram agarose powder for 1% gel concentration 0.3 gram agarose powder for 1.5% gel concentration 0.4 gram agarose powder for 2% gel concentration 0.5 gram agarose powder for 2.5% gel concentration