2. Agarose Gel Electrophoresis Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry,molecular biology, genetics, and Clinical chemistry to separate a mixed population of macromolecules such as DNA or Proteins in a matrix of agarose, One of the two main components of agar. The protein may be separated by charge and or size (Isoelecteric focusing agarose electrophoresis is essentially size independent) and the DNA and RNA fragments by length. 1.Gel electrophoresis is a technique used to separate DNA fragments according to their size. 2.DNA sample are loaded into wells(indentations) at one end of the gel, and an electric current is applied to pull them through the gel. 3.DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass,small fragments move through the gel faster than large ones. 4.When a gel is stained with a DNA –binding dye, the DNA fragments can be seen as bands,each representing a group of same sized DNA fragments. 5.All DNA molecules have the same amount of charge per mass. Because of this gel electrophoresis of DNA fragments separates them based on size only. Using electrophesis, we can see how many different DNA fragments are present in a sample and how large they are relative to one another. 6.We can also determine the absolute size of a piece of DNA by examining it next to a standard “ yard stick” made up of DNA fragments of known sizes. 7.A well defined “ line” of DNA on a gel is called a “ Band". Each band contains a large number of DNA fragments of the same size that have all traveled as a group to the same position.

3. 8. A single DNA fragments (or even a small group of DNA fragment) would not be visible by it self on a gel. 9. The bright band on the gel above is roughly 700 base pairs(bp) in size. How to prepare the Agarose Gel Requirements: 1. A horizontal electrophoresis apparatus. 2. A DC power source 3. A micropipette or Transfer pipette 4. agarose Power 5. Electrophoresis buffer 6. Sample for the experiments. Procedure: Dilute your sample with concentration buffer with deionized water. Step1: Mix agarose powder with your diluted buffer solution in a 250 ml flask. Step2: Dissolve the agarose powder by boiling the solution (Microwave the solution on high for one minutes)

4. Carefully removed the flask from the microwave and mix by swirling (Beaker rotating) Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (The solution should look clear like water) Step 4 : Cool agarose gel to 60◦C with care full swirling to promote the even dis sipation of heat. Step 5: While the agarose is cooling, SEAL the ends of the gel casting tray with the rubber end caps. Place the well template into the appropriate match Step 6: Pour cooled agarose solution the prepared gel casting tray. The gel should solidify within 20 minutes. The gel will stiffen and become less transparent as it solidifies. Step7: Remove the end caps and comb

5. Take Particular care when removing the comb and end caps to prevent damage to the gel. And Now you have your gel prepared and ready for electrophoresis ! Now continue to ‘ performing ‘ electrophoresis. Refer to table A for the specific amount according to your casting trays size ! Size of the Gel casting tray Amount of Agarose Concentra tion of buffer(50u L) Dilute water Total Volume 7 x 7 cm0.23g0.6 ml29.4 ml30 ml 7 x 10cm0.39 g1.0ml49.0 ml50 ml 7 x 14 cm0.46 g1.2 ml58.8 ml60 ml

6. DNA + Protein Interaction DNA: Thaw it in ice(slowly) don’t thaw with palms Take 50 ml of DNA + 450 ml of autoclaved milli Q water Sonication @ 10s ON,20s OFF(for 1 minutes) Measure OD 260(UV-visible ) note the value Load on Agarose gel (5 ml.Max ; 2ml Min) Check how many base pair band is seen. after that calculate the concentration by OD 260 = 1……….50ug/ml vs DNA OD 260 =………… ˀ vs DNA If no. of base pair is known then we can calculate the concentration in …………(Morality).

7. Notes 1.Agarose gel electrophoresis is most suitable for separation of DNAs and RNAs in the range of 100bp to 15kb. 2.DNA already has uniform negative charge. If you recall DNA structure from our last class, two nucleotides are connected together by a phosphodiester bond which gives one negative charges. 3.Here, we are ignoring one negative charge due to phosphate at 5’ end as this is very small compare to net charge provided by phosophodiesterbonds in a polynucleotide.

8. There are several physical methods available for separating nucleic acid (DNAand RNA)based on its size. Gel electrophoresis is a separation technique which is purely based on charge and size. Agarose gel electrophoresis is eventually one of the traditional methods of separating and analyzing nucleic acid. In this method, gel made from agarose acts as a separating medium. Agarose which is linear polymer agarose is a polysaccharide, whose monomeric unit is a disaccharide of D-galactose and 3,6-anhydro-L-galactopyranose. Running a Gel: Load your DNA samples in corresponding wells in the gel. Remember that DNA is negatively charged and runs towards the positive electrode. The gel wells should be nearer towards black electrode and farther from red electrode (DNA should run to the red end). Turn on the power to run the gel with the voltage set at 60-80 volts for 40 minutes for small gels and 90-100 volts for 1- 2 hours for larger gels. Agarose gels run at high voltage may result in melting the gel and distortion of bands. One can confirm that the gel is running by checking for bubbles from the electrodes. Switch off the power at the end of the run.

10. Analyzing a Gel: