IGEM:Peking/2007/Switch: DNA electrophoresis

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(含不同浓度琼脂糖的凝胶的分离范围)
Current revision (08:11, 20 October 2007) (view source)
(method of making gel)
 
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==DNA 电泳==
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==DNA eletrophoresis==
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以下指0.5cm加样孔。2ng为检出下线,500ng以上拖尾。
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0.5cm slot, 2ng is the bottom line for testing.
===Markers===
===Markers===
Lambda DNA/EcoRI+HindIII Marker Fermentas
Lambda DNA/EcoRI+HindIII Marker Fermentas
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0.5ug/uL,3uL每次
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0.5ug/uL,3uL
    
    
Transgen 100bp DNA Ladder
Transgen 100bp DNA Ladder
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5uL 每次
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5uL
   
   
Transgen 1kb DNA Ladder
Transgen 1kb DNA Ladder
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5uL 每次
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5uL
    
    
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===含不同浓度琼脂糖的凝胶的分离范围===
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===Agarose concentrations===
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琼脂糖含量[%(w/v)]:  线状DNA有效分离范围[kb]
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agarose concentration[%(w/v)]:  linear DNA effective distinguishment[kb]
0.3         :            5-60
0.3         :            5-60
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2.0           :            0.1-2
2.0           :            0.1-2
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===电泳时间===
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===Timing===
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以要观测的DNA明显与其他长度的DNA分开为宜,相应长度范围的Marker应得到分离清晰的图像。EB与DNA电泳方向相反,电泳时间过长会使EB迁移出凝胶,此时应将凝胶浸于1×TAE+EB溶液30min后成像。
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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.
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Under such situation, the gel should put in 1×TAE+EB solution for 30min before imaging.
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===Loading Buffer的选取===
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===Loading Buffer===
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在0.5-1.4%的琼脂糖中,溴酚兰的移动速度相当于300bp的双链线状DNA,二甲苯青FF的移动速度相当于4kb的双链线状DNA。以染料不遮挡要观测的DNA为宜。
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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.
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===溶液配方===
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===solutions===
====50×TAE 1L====
====50×TAE 1L====
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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)
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定容至1L
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constant volume to 1L
====1×TAE +EB 1L====
====1×TAE +EB 1L====
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====10×Loading Buffer 50mL====
====10×Loading Buffer 50mL====
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溴酚兰/二甲苯青FF 20g 40% 溴酚兰/二甲苯青FF
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20g 40% bromophenol blue
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甘油 25mL
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glycerol 25mL
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50% 甘油
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constant volume to 50mL
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定容至50mL
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===method of making gel===
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===倒胶方法===
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Use 1×TAE+EB to make the gel, 1×TAE as the eletrophoresis buffer
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用1×TAE+EB配胶,1×TAE为电泳缓冲液
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The agarose should be shaken up and pour out as the gel
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琼脂糖加热后要摇匀,待稍冷却后倒胶
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the slot lies at the negative electrode
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加样孔在负极
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the voltage should be remained constant during electrophoresis
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恒压电泳,电流不可超过电泳仪量程
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===EB pollution===
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只使用一个电泳槽时,确保空闲的电泳槽断开
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EB is a strong carcinogen.
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EB污染!!
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In EB polluted area we should use gloves to touch anything.
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EB为致癌物质。
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EB污染区内任何物品应戴手套触摸。
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严禁用触摸过含EB物质的手套触摸EB污染区以外的任何物品。
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严禁将含有EB的物品拿出EB污染区以外。
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电泳液单独回收,严禁倒入下水道内。
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EB污染区和含EB的容器使用后请自己清洗整理干净。
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EB污染区以外范围需尽快小心清理干净。如触摸到EB,应用大量水冲洗。
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EB污染台微波炉上表面以上为非污染区,UVP仅暗箱内部紫外部分为污染区。
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Current revision

Contents

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.

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