IGEM:Peking/2007/Switch: DNA electrophoresis

DNA eletrophoresis
0.5cm slot, 2ng is the bottom line for testing.

Markers
Lambda DNA/EcoRI+HindIII Marker Fermentas

0.5ug/uL，3uL Transgen 100bp DNA Ladder

5uL Transgen 1kb DNA Ladder

5uL

Agarose concentrations
agarose concentration[%(w/v)]: linear DNA effective distinguishment[kb]

0.3	        :             5-60

0.6	         :            1-20

0.7	         :            0.8-10 0.9	          :            0.5-7

1.2	         :            0.4-6

1.5	         :            0.2-3

2.0	         :            0.1-2

Timing
distinguish different size of DNA. EB moves opposite direction to DNA and will run out of the gel if the time for electrophoresis is too long. Under such situation, the gel should put in 1×TAE+EB solution for 30min before imaging.

Loading Buffer
In 0.5-1.4% agarose，the speed of bromophenol blue is the same as 300bp double strand linear DNA，the speed of Xylene blue is the same as 4kb double strand linear DNA.

50×TAE	1L
Tris	242g

2M Tris-AC

HAC	57.1mL

0.5M EDTA（pH 8.0）100mL	,0.05M EDTA（pH 8.0）

constant volume to 1L

1×TAE	+EB	1L
50×TAE		20mL

0.04M Tris-AC+0.001M EDTA （pH 8.0）

10mg/mL EB	50uL	0.5ug/mL

H2O		980mL

10×Loading Buffer	50mL
20g 40% bromophenol blue

glycerol			25mL

constant volume to 50mL

method of making gel
Use 1×TAE+EB to make the gel, 1×TAE as the eletrophoresis buffer

The agarose should be shaken up and pour out as the gel

the slot lies at the negative electrode

the voltage should be remained constant during electrophoresis

EB pollution
EB is a strong carcinogen.

In EB polluted area we should use gloves to touch anything.