GEL ELECTROPHORESIS - mcdb凝膠電泳- MCDB

1、.HC70AL SPRING 2004 PROFESSOR BOB GOLDBERGGEL ELECTROPHORESISReference: Sambrook et al. (1989). Molecular Cloning Manual.Solutions Needed:Ø AgaroseØ 1X TAE bufferØ 10mg/mL Ethidium Bromide (EtBr)Ø 6X Loading Dye containing xylene cyanol and bromophenol blue dyesApparatus Needed:&

2、#216; Gel castØ Gel boxØ CablesØ Gel combØ Plastic (Saran) wrapGEL ELECTROPHORESIS1. Measure out X grams of agarose (powder) depending on the final percentage of agarose in the gel.Example: If you want to make a 1% agarose gel (1 g/100 mL, w/v), weigh out 1g of agarose for 100 mL

3、 of agarose solution2. Carefully, put the agarose in a 250-mL Erlenmeyer flask.3. Measure out 100 mL of 1X TAE buffer using a plastic or glass graduated cylinder.4. Add 100 mL of 1X TAE buffer into the flask in step 2.5. Cover the flask with a piece of plastic wrap. Poke 3-4 holes on the plastic wra

4、p using a pointed end of a pencil or pen (note: the holes allow the steam to escape during microwaving in step 6 below). Swirl the solution to break up any lumps of agarose granules.6. Microwave the solution for about 2 minutes or until the agarose granules have completely melted.Ø Be careful w

5、ith the flask. The solution gets very hot.Ø Constantly watch over the solution because when it starts boiling, it might overflow.Ø Swirl gently the solution several times while microwaving to help melt agarose evenly.Ø Once the agarose has melted completely, the solution is clear.

6、6; Once the agarose has melted completely, the solution is clear.7. Cool down the agarose solution for at least 30 min in a 55ºC water bath.8. While the agarose solution is cooling, prepare the gel cast with the appropriate comb. Ø The comb depends on the number of PCR samples. For example

7、, if there are 31 samples, then a 40-tooth comb is needed.Ø Remember to add two more wells to the number of wells needed for the samples. These two wells will be for loading 1kb DNA ladder in the first and the last wells.9. Add 2mL of loading dye in newly labeled microcentrifuge tubes for each

8、PCR sample. The tubes are labeled according to the samples.10. Aliquot 10 mL of PCR sample into microcentrifuge tubes.11. After the agarose solution has been cooled down, add to it 5 mL of Ethidium Bromide (EtBr) and swirl to mix.12. Pour the agarose/EtBr solution into the gel cast. Wait for 30 min

9、for the agarose solution to solidify.13. Pour 600 mL of 1X TAE buffer into the gel box.14. After the agarose has solidified into a gel, take out the comb gently and put the gel in the gel box.15. Load the samples starting from the second well. Note: the first well will contain the 1kb DNA ladder.16.

10、 Load 10 mL of diluted 1kb DNA ladder solution (50 ng of DNA/mL) into the first and the last wells.17. Add 10 mL of 10 mg/mL of EtBr to the anode (positively charged) of the gel box. (The anode is the opposite side from wells)Ø Ethidium bromide is positively charged. Therefore, it migrates towa

11、rds the negative end of the gel box from anode to cathode. (Opposite direction from DNA migration)Ø Remember that DNA is negatively charged; so, it migrates to the positive end of the gel. (DNA migrates from cathode to anode)18. Put the lid of the gel box on the gel box and connect the electrod

12、es to the power supply (RED to RED and BLACK to BLACK).19. Turn on the voltage of the power supply to 94 volts for ONE gel (or 130 volts for TWO gels connected to the same power supply) and wait for 2 hrs or until the front dye (bromophenol blue or BPB) has migrated two-thirds of the gel length. Not

13、e: the amount of voltage and duration of running the gel(s) depend on types of the power supply and gel-electropheris systems made by different manufacturers (such as Bio-Rad, Owls System, or Invitrogen). For example, it would take 1.5 hours or 2.5 hours for the BPB dye migrating to two-thirds of th

14、e gel for a Bio-Rad or Owls gel-electrophoresis systems, respectively, at 78 volts.20. Turn off the power supply.21. Remove the lid of the gel box. Put the gel on its gel cast into a small plastic container and bring the container to room 2828.Caution: it is a MUST to put the gel into a plastic cont

15、ainer so that the gel would NOT slide off the gel cast, fall on the floor and be broken into pieces while walking to a different room (2828) for taking a picture of the gel.22. Take a picture of the gel using the BioRad Gel Document System in room 2828. 23. Label the picture using a text program of the Gel Document System (your TA will show you how).24. Print out the picture.25. (Optional) Label the picture by: Ø putting a piece of wh