Strategies for electrophoretic separations

Several parameters must be considered before performing an electrophoretic separation of HMW DNA. The separation of large DNA molecules in agarose gels is affected by the agarose concentration, buffer concentration, temperature, pulse times, voltage, and total electrophoresis running time.

• The agarose concentration determines the size range of DNA molecules separated, and the sharpness or tightness of the bands. Agarose concentrations of 1 % are useful in separating DNA molecules up to 3 Mb in size.

• Tris-borate buffer (TBE), at a concentration of 0.5 X and chilled to 14 °C, is recommended for use in pulse-field electrophoresis.

• The migration rate of DNA molecules through agarose gel is determined by voltage, pulse time, and running time. The DNA migration velocity increases with increasing voltage and temperature, and decreases with increasing agarose concentration. Greater migration is accompanied by

Table 2. Run parameters

DNA size (Mb)

<0.1

0.1-0.2

2-4

>4

Agarose (%)

1.5

1-1.5

0.8-1

0.5-0.8

TBE buffer

0.5 X

0.5 X

0.5 X

0.5 X

Temperature (°C)

14

14

14

14

Voltage (V)

200

150-200

125-175

40-75

Pulse time (s)

1-10

1-10

1-10

10-60 min

Run time (h)

6-12

18-24

24-72

3-6 days

Table 3. Examples for pulse mode

DNA size (Mb)

0.2-23 kb

0.05-1

0.2-2.2

1-5

3.5-5.7

Mode

2

2

3

3

1

Agarose (%)

1.5

1

1

0.8

0.6

Pulse time (s)

1-4

50-90

60-90

120-240

60 min

Run time (h)

5

24

15-19

24-36

160

Voltage (V)

200

200

200

150

50

decreased band sharpness. Increased band sharpness requires longer run times at low voltage. Therefore, a compromise in voltage, temperature, and agarose concentration should be used to give the maximum allowable band sharpness within a reasonable run time. Table 2 gives a general starting point for parameters to separate DNA molecules in the various size categories.

There are three common pulse modes used in CHEF analysis (see Table 3). Mode 1 is a single pulse time for the duration of the run. Mode 2 is an abrupt change in pulse time, with one pulse setting for the first part of the run and a second pulse setting for the remainder of the run. Mode 3 is a gradual linear change of the pulse time from the initial to the final pulse time.

0 0

Post a comment