Agarose Gel Electrophoresis

Small Gel:

• Weigh out 250 mg agarose, place in 125 ml flask
• Add 25 ml DI, 2.5 ml 10X TAE buffer
• Put flask in microwave for 25 s or until it just boils. Repeat using 5 s pulses until agarose melts and solution is completely clear. Hold the solution up to the light and make sure all chunks are melted, yet do NOT let it boil over in the microwave.
• Set up the casting tray by taping each end with autoclave tape and put the comb near one end.
• Let the agarose cool until it is warm to the touch.
• Pour into casting tray (with comb) that has been sealed at both ends with tape. Make sure comb is facing toward the top end of the gel.
• Let solidify 30 min.
• Make tank buffer: 225 ml DI, 25 ml 10 x TAE
• Put gel in minisub reservoir and cover with tank buffer. Wells should be facing the black pole. DNA runs towards the red (+).
• Mix samples to be run a total of 10-12 ul, including 2 ul of loading buffer. Make sure you use a phage lambda (Hind III digest) as molecular weight standards.
• Mix the samples well and carefully load into each well, without overflowing the wells.
• Put plexiglass lid on gel apparatus and plug banana jacks into the powersource, black with black and red with red.
• Turn on power source and adjust volts to 50-60 volts (4.5-5 volts/cm) Make sure bubbles are coming up at electrodes and gel is running correctly.
• Let gel run until the tracking dye is 2 cm from bottom (2-3 hr).
• Photograph gel

     Base Pairs	Daltons	
     23130	15x106	
     9419	6.12 x106	
     6557	4.26 x106	
     4317**	2.84 x106	
     2322	1.51 x106	
     2028	1.32 x106	
     546	0.37 x106	
     **125		

• *Usually not visible