Running a 1% agarose gel 1. Gently remove the comb(s) from the pre-poured gel. The gels have ethidium bromide in them- BE CAREFUL and wear gloves!! 2. You should load 10 ul of cDNA/PCR sample + 5 ul of dye. Also, in a separate late, load 10 ul of marker (NEB 1 kb marker)—this tells us what the size of our cDNA is (see figure to the right). The smaller the fragment of DNA, the faster it runs and the smaller DNA fragments will be toward the bottom (see the marker scale again). Cut a piece of Parafilm and tape it to your desk or the flat side of a microfuge tube rack- see below. Pipette the loading dye on the Parafilm, one dot per sample. etc. sample 2 sample 1 marker The loading dye is made up at a 6x concentration, but if you are working with small volumes, it is OK to exceed a 1x final concentration. For example: 5 ul dye + 5 ul sample or 5 ul dye + 2 ul sample or 2 ul dye + 10 ul sample Pipette your samples into the dye spots. Set your pipette to the total volume. Pipette up and down to mix the dye + sample. _ 3. Transfer each sample to the slot left after removing the comb. This is “loading the gel” with your samples (be sure your gels are in sample buffer with glycerol and dye)—see figure to the right. Record the order of your samples in your notebook (left to right). 4. Run the gel at 150 Volts (current should flow negative to positive – black on top, red on bottom). This is constant voltage (you can also run gels with constant current). Proteins and DNA/RNA are negative, so put the red positive electrode (anode) at the bottom of the gel box. PHOTO: Gel box on left, power supply on right, comb in front. + 1 Photography of the gel. 5. 5. Carry your gel (in the gel tray) to the UV light box. USE YOUR gloved fingers to hold the gel in place or it will slide off!! Be sure not to drip buffer on the floor and don’t let the gel fall to the floor (they are fragile). EtBr IS A CARCINOGEN – WEAR GLOVES – CLEAN UP ANY SPILLS IMMEDIATELY – FROM NOW ON TREAT THE GEL AS A BIOHAZARD AND WEAR GLOVES 6. Put on your UV glasses or face shield. Be aware of others around you, this is a powerful UV light. 7. Turn on the UV light and view your gel. 8. If it looks good, take a picture with the digital camera. Look for the slot opposite the handle and find the camera settings. Start off with settings of an F stop of 16 (this is how wide open the “lens” of the camera ismore open, better pictures with lower light) and a 1 sec exposure. You may have to open the lens more with a setting of F 8.Photo below shows time of exposure (seconds) on the top and the F stop lens opening on the bottom. 2 Appendix: Pouring Gels: 1. Measure 60 mL of 1X buffer (TBE) in a 500 mL Erlenmeyer flask. 2. Add 0.6 g of agarose. 3. Put a weigh boat on top of flask. 4. Heat in microwave until boiling. BE CAREFUL – ITS HOT – STEAM IS HOT KNOCK OFF WEIGH BOAT LID BEFORE REMOVING FLASK FROM MICROWAVE 5. Swirl and re-heat until the solution is clear and you cannot see anything floating. 6. Let solution cool so that glass is warm to the touch but not burning hot. Alternatively, the flask can be placed in a 55 C water bath to cool and keep the agarose melted. 7. Add 6 uL of 10 mg/mL Ethidium Bromide to flask and swirl. EtBr IS A CARCINOGEN – WEAR GLOVES – CLEAN UP ANY SPILLS IMMEDIATELY – FROM NOW ON TREAT THE GEL AS A BIOHAZARD AND WEAR GLOVES 8. Set up your gel apparatus. Be sure ends are blocked off. Use the appropriate size combs – be sure you have enough wells for all your samples plus markers and controls. 9. Pour the melted, but cooled agarose. Let the gel solidify. It will become opaque looking. 10. Turn the gel so that the wells are near the black electrode (the negative end). 11. Pour enough 1X buffer (TBE) to just cover the top of the gel and fill the side reservoirs. Too much buffer over the top will result in a longer running time. 3