1 / 22

GEL ELECTROPHORESIS

HOW TO POUR A 0.7% AGAROSE GEL. Pour 50 ml of 1X TBE buffer (in the graduated cylinders) into an ehrlenmeyer flask or small beaker.Add 0.35 g (in the Dixie cups) of agarose. Swirl gently. Let sit 1-2 minutes.Loosely cover the flask with foil if desired. Heat the agarose mixture on a hot plate until it just begins to boil.Swirl gently (no air bubbles!). Let cool down to comfortable

ike
Télécharger la présentation

GEL ELECTROPHORESIS

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

    1. GEL ELECTROPHORESIS Standard Operating Procedure

    2. HOW TO POUR A 0.7% AGAROSE GEL Pour 50 ml of 1X TBE buffer (in the graduated cylinders) into an ehrlenmeyer flask or small beaker. Add 0.35 g (in the Dixie cups) of agarose. Swirl gently. Let sit 1-2 minutes. Loosely cover the flask with foil if desired. Heat the agarose mixture on a hot plate until it just begins to boil. Swirl gently (no air bubbles!). Let cool down to comfortable baby bottle temperature.

    3. Tightly seal the ends of the gel tray with lab tape. If desired, place the tray sideways in the dry gel box for additional sealing pressure. Pour the warm (NOT hot) agarose into the tray. Insert the comb. Let solidify 20-30 minutes. HOW TO POUR A 0.7% AGAROSE GEL

    4. Pick the correct micropipet for your volume (P20). Slowly dial until it reads 10.0 ul. Press the micropipet into a fresh yellow tip in the box to attach the tip. Using your thumb, slowly depress the piston until you feel the first stop. HOW TO USE A MICROPIPET

    5. Working @ eye level, insert the tip just below the surface of the colored water in the beaker. Slowly release the piston to draw 10 ul into the tip. Withdraw the tip from the water. Lightly touch the tip against the wall of the receiving vessel or the wax paper, then slowly depress the piston past the first stop. HOW TO USE A MICROPIPET

    6. HOW TO LOAD AN AGAROSE GEL Remove the lab tape from the gel tray. Insert the gel tray into the gel box with the wells closest to the black lead. Pour enough 1X TBE buffer into the gel tray to barely cover the gel. Carefully remove the comb.

    7. Record which samples are to go into which lanes. Using a micropipet and a disposable tip, withdraw 10 ul of sample from the microcentrifuge tube. Keeping the micropipet steady, slowly insert the tip half way into the desired well. Carefully check tip position by feeling for the walls of the well. Slowly depress the plunger past the first stop to deliver the sample to the well. Do not release the piston. Wait 1 second before pulling the tip out of the gel box. Release the piston and eject the tip. HOW TO LOAD AN AGAROSE GEL

    8. Close the gel box with the cover. Double-check that the black lead is near the wells. Hook the leads up to the power supply. Turn on the power supply to read 90-100 V for each of the channels that are in use. Check to make sure bubbles are coming from the wire electrodes in the buffer. This means it is working. Run until the blue dye is 2/3 to 3/4 of the way down the gel. HOW TO RUN AN AGAROSE GEL

    9. HOW TO STOP AND STAIN AN AGAROSE GEL Put on gloves and prepare a 100X FastBlast stain bath. Turn the power supply down to 0 V for each channel that is in use. Turn off the power. Remove the gel box cover. Reach into the gel box and remove the gel tray (careful so the gel does not fall off the tray).

    10. Carefully slide the gel off the gel tray into the bath. Let sit for a maximum of 3 minutes. Pour off the dye into another tray, then add warm tap water. Agitate gently for 1 minute. Pour off the water down the sink. Add fresh warm tap water. Agitate gently. Repeat this step a few more times until the water runs clear. Let the gel sit at least 15 minutes in the last water bath. View the gel on white paper or on a white light box. Take a digital picture to print if measurements are desired. HOW TO STOP AND STAIN AN AGAROSE GEL

    11. HOW TO GENERATE A STANDARD CURVE On the printed gel picture, draw a line where the wells are. For each of the bands in the DNA size marker lane, measure the distance traveled from the well in mm. On 3-cycle semi-log paper, plot the known size of each band (in kb) on the log-scale (Y-axis) vs. the distance migrated (in mm) on the regular scale (X-axis). Draw a best-fit straight line through the data points. This is your standard curve for this particular gel.

    13. HOW TO RUN A VIRTUAL GEL ONLINE http://tools.neb.com/NEBcutter2/index.php

More Related