IGEM:Peking/2007/Switch: DNA electrophoresis

From OpenWetWare

< IGEM:Peking | 2007
Revision as of 07:11, 20 October 2007 by Chen Chongyi (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

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.

Personal tools