AGAROSE GEL PROCEDURE

1.  Assemble the gel casting tray and comb. The comb should not touch the bottom of the tray.

2.  Add  agarose (Table 1) to 65 mL 0.5x TBE buffer.  Using a microwave, melt the agarose solution.

Table 1.  Preparation of agarose solutions

Gel Composition (%)
Standard Agarose (g)
0.6
0.4
0.8
0.5
1.0
0.65
1.2
0.8
1.5
0.98

3.  When the agarose solution has cooled to about 50°C, pour solution directly into the casting tray, ensuring that no bubbles get into the gel.  (rinse the flask immediately)


4.  Allow the gel to cool. It will solidify and become slightly opaque within 20 to 30 minutes.  Remove black end pieces.


5. Submerge the gel by adding approximately 1L of 0.5X TBE running buffer to cover the gel by about a half a centimeter.


7.  Carefully remove the comb by lifting it gently at one end, tilting the comb as it comes out. Pulling the comb straight up creates a vacuum in the wells, which tends to lift the whole gel out of the tray. Ensure that the wells are submerged and filled with buffer.

8.  Prepare the DNA samples for loading using glycerol loading buffer.

9.  Load a maximum of 10 mcL of each sample into individual wells with a gel loading tip and a P-20 Pipetman.





10.  Once all the samples are loaded place the cover on the gel apparatus. Connect the leads so that the red (positive) lead is at the end of the gel to which the DNA will migrate and the black (negative) lead is at the end of the gel containing the wells. Turn on the power supply and set according to the instructor's guidelines. Check the gel after a few minutes. If the dye migrates in the wrong direction, turn off the power supply and switch the leads. Run at a constant voltage of 200 volts. CAUTION: DO NOT REMOVE THE LID FROM THE GEL APPARATUS WITHOUT DISCONNECTING THE POWER LEADS. ELECTRICAL SHOCK MAY RESULT.

11.  When the blue tracking dye (which runs in these gels along with a DNA fragment of about 200-400 bp) has migrated about 75% of the distance to the end of the gel (usually within 60-90 minutes), turn off the power supply and disconnect the power leads.

Caution, the gel is very slippery!!

12.  Carefully transfer the gel into a plastic dish and add enough 1X SYBR Gold staining solution to cover the gel.  Set in a dark drawer for 30 minutes.

13.  Visualize the DNA with UV light on the Lumi-Imager.

13.  Dispose of the gel into the trash.

14.  Rinse the light box and tray with dI water and dry it with paper towels.
 

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