DNA agarose gel electrophoresis

This presentation is intended to help different persons to get knowledge and skills on agarose gel electrophoresis.

1. Molecular biology DNA AGAROSE GEL ELECTROPHORESIS Prepared by E. NSENGIYUMVA, BSc Lab technician University of Rwanda College of Medicine and Health Biomedical Laboratory Sciences

2. INTRODUCTION Electrophoresis is a technique of separating molecules basing on their electrical charges, where positively and negatively charged molecules migrate to cathode (negative) and anode (positive) poles respectively. Molecules with the same charges, are further separated according to their size where larger molecules migrate slower than small ones.

3. INTRODUCTION(Cont.) Agarose gel electrophoresis is a routinely used method for separating DNA or RNA. Nucleic acid molecules are size separated by the aid of electric field where negatively charged molecules migrate toward anode pole. The agarose gel electrophoresis is the most used and effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 Kb.

4. Introduction (CONT;) Agarose gel consists of microscopic pores that act as a molecular sieve which separates molecules based upon charge, size, and shape. These characteristics together with buffer conditions, gel concentration and voltage affect the mobility of molecules in gels.

5. INTRODUCTION(Cont. Application of DNA agarose gel electrophoresis DNA fragment size estimation DNA purification Genetic fingerprinting PCR product analysis

6. PRINCIPLE DNA sample is loaded into pre-cast wells in the agarose gel and a current applied. The phosphate backbone of the DNA (and RNA) molecules is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio. DNA molecules are separated by size; the smaller molecules migrate faster than large molecules

7. Materials and reagents Materials Gel casting tray Gel combs Electrophoresis chamber Electrophoresis power supply Tank cover Electrical leads UV transilluminator Chemical Balance Erlenmeyer Flask Oven or heater Personal protective equipments Timer Reagents 50x TAE Buffer Agarose gel powder DNA Ladder Ethidium Bromide or SBR safe Gel loading dye Distilled water

8. 1X TAE buffer preparation 1x TAE Buffer is used in agarose gel preparation and for running gel electrophoresis Prepared from 50x TAE Buffer Add 20 mL of 50x TAE stock solution to a 1 L Duran bottle. Add 980 mL of distilled water . Mix the solution by shaking .

9. Agarose gel preparation Percent Agarose Gel (W/V) DNA Size Resolution (kb=1000) 0.5% 1 kb to 30 kb 0.7% 800 bp to 12 kb 1.0% 500 bp to 10 kb 1.2% 400 bp to 7 kb 1.5% 200 bp to 3 kb 2.0% 50 bp to 2 kb Agarose gel is prepared depending on your DNA size

10. 1% Gel preparation Measure out 25 mL of 1X TAE using a graduated cylinder Pour into a 100 mL Erlenmeyer flask. Measure 0.25 g of agarose and add to flask to produce a ~ 1.0% w/v agarose gel. Heat the flask using a microwave to melt the agarose. Swirl and mix the agarose. Let cool at 60 C for 3 minutes on the bench. Add 2.0 µL (1/10000) of Ethidium Bromide (EtBr ) [Carcinogenic, PPE must be worn) or SYBR Safe and swirl . Pour onto assembled gel tray with comb. Use a pipette tip to move any air bubbles to the side or the bottom of the tray. Agarose will solidify after a few minutes and turn translucent, glossy.

11. Sample loading Place tray with solidified gel into the gel box, and gently remove the comb from the gel . Pour TAE buffer into the gel box, the gel must be covered with buffer. Carefully add 1 µL of loading dye to each ~ 10 µL DNA sample. Carefully pipette 7 µL of 1kb ladder into the first lane of the gel. Load 15 µL each of the samples into separate wells by pipetting, as described above .

12. Running the gel Place the lid of the gel box properly (black with black, and red with red). Run the gel at 100 Volts for about 45 minutes or until the blue dye has run about 1/2 to 2/3 across the gel. Turn off the current and remove leads.

13. Gel documentation Remove the lid of the gel box. Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place the gel tray on paper towels to absorb any extra running buffer. Remove the gel from the gel tray and put in the gel documentation machine Expose the gel to UV light. This is most commonly done using a gel documentation system. DNA bands should show up as orange fluorescent bands. Take a picture of the gel and store on flash disk or in computer for gel fragment size observation. Properly dispose of the gel after gel documentation or DNA purification.

14. Agarose Gel Documentation

15. References Lee, P. Y. et al. (2012) ‘Agarose gel electrophoresis for the separation of DNA fragments’, Journal of Visualized Experiments , (62), pp. 1–5. doi : 10.3791/3923. Magdeldin , S. (2012) Gel Electrophoresis - Principles and Basics , Gel Electrophoresis - Principles and Basics . doi : 10.5772/2205.

16. THANKS