112
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND BIRGIT WEISS
Table 2. Continuos
Pulse Time( s)
Run Time (h)
Valtage (V)
Separation Range
(kb)
10- 30
24
180
10 - 300
20- 80
42
140
10- 700
20 - 100
42
140
10 - 1150
40 - 150"
34
140
150 - 300"
8
140
10 - 1500
• These two pulse time gradients are run consecutively
Comments
Optimizing running conditions
To optimize conditions for separation ofDNA fragments in the appropriate
size range, several parameters can be changed:
• Pulse time
The lower the selected pulse time, the smaller the resolved molecules
(Table 1 ). Separationrange may differ within a given pulse time, depending on the gel box and running system used.
• Voltage/current
Generally, gels are run at constant valtage (10 V/ern). For separation of
DNA larger than 2 Mbp, valtage must be lowered because of trapping
effects. The higher the valtage gradient, the shorter the switching interval
for separating the same range of size markers. Current will increase with
running time.
• Concentration of agarose in gel
Agarose concentration between 0.8 and 1% are optimal for most purposes. Use of 0.7% rather than 1% agarase can speed up lang gel
runs but decreases the resolution and broadens the band width.
• Temperature
Constant temperaturein the gelbox is very important; preferred temperatures are between 10-15°C, cooled by a Kryostat.
• Run time
Usually, running times are between 16-48 h; separation of fragments larger than 2 Mbp require running times langer than two days.
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND BIRGIT WEISS
Table 2. Continuos
Pulse Time( s)
Run Time (h)
Valtage (V)
Separation Range
(kb)
10- 30
24
180
10 - 300
20- 80
42
140
10- 700
20 - 100
42
140
10 - 1150
40 - 150"
34
140
150 - 300"
8
140
10 - 1500
• These two pulse time gradients are run consecutively
Comments
Optimizing running conditions
To optimize conditions for separation ofDNA fragments in the appropriate
size range, several parameters can be changed:
• Pulse time
The lower the selected pulse time, the smaller the resolved molecules
(Table 1 ). Separationrange may differ within a given pulse time, depending on the gel box and running system used.
• Voltage/current
Generally, gels are run at constant valtage (10 V/ern). For separation of
DNA larger than 2 Mbp, valtage must be lowered because of trapping
effects. The higher the valtage gradient, the shorter the switching interval
for separating the same range of size markers. Current will increase with
running time.
• Concentration of agarose in gel
Agarose concentration between 0.8 and 1% are optimal for most purposes. Use of 0.7% rather than 1% agarase can speed up lang gel
runs but decreases the resolution and broadens the band width.
• Temperature
Constant temperaturein the gelbox is very important; preferred temperatures are between 10-15°C, cooled by a Kryostat.
• Run time
Usually, running times are between 16-48 h; separation of fragments larger than 2 Mbp require running times langer than two days.