IGEM:Peking/2007/Switch: DNA electrophoresis: Difference between revisions
Chen Chongyi (talk | contribs) |
Chen Chongyi (talk | contribs) |
||
| (2 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
==DNA | ==DNA eletrophoresis== | ||
0.5cm slot, 2ng is the bottom line for testing. | |||
===Markers=== | ===Markers=== | ||
Lambda DNA/EcoRI+HindIII Marker Fermentas | Lambda DNA/EcoRI+HindIII Marker Fermentas | ||
0.5ug/ | 0.5ug/uL，3uL | ||
Transgen 100bp DNA Ladder | Transgen 100bp DNA Ladder | ||
5uL | 5uL | ||
Transgen 1kb DNA Ladder | Transgen 1kb DNA Ladder | ||
5uL | 5uL | ||
=== | ===Agarose concentrations=== | ||
agarose concentration[%(w/v)]: linear DNA effective distinguishment[kb] | |||
0.3 | 0.3 : 5-60 | ||
0.6 | 0.6 : 1-20 | ||
0.7 | 0.7 : 0.8-10 | ||
0.9 | 0.9 : 0.5-7 | ||
1.2 | 1.2 : 0.4-6 | ||
1.5 | 1.5 : 0.2-3 | ||
2.0 | 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 | ===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. | |||
=== | ===solutions=== | ||
====50×TAE 1L==== | ====50×TAE 1L==== | ||
| Line 50: | Line 51: | ||
0.5M EDTA（pH 8.0）100mL ,0.05M EDTA（pH 8.0） | 0.5M EDTA（pH 8.0）100mL ,0.05M EDTA（pH 8.0） | ||
constant volume to 1L | |||
====1×TAE +EB 1L==== | ====1×TAE +EB 1L==== | ||
| Line 64: | Line 65: | ||
====10×Loading Buffer 50mL==== | ====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. | |||
Latest revision as of 05:11, 20 October 2007
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.
solutions
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.
