Molecular Techniques and Methods
Removal of Unincorporated dNTPs from
Labeling Reaction Using Home-Made Apparatus
Copy Right © 2001/ Institute of Molecular Development LLC
INTRODUCTION
MATERIALS AND SOLUTIONS
Color Indicator Solution (1 ml)
2.5% Blue Dextran
0.5%
Bromo Phenol Blue
Dissolve in deionized H
2
O
Bio-Gel P-60 Suspension (
Bio-Rad
)
Bio-Gel P-60 ------------------------------------- 1 g
Distilled H
2
O ------------------------------------- 20 ml
Allow to swell Bio-Gel P-60
overnight
before use.
PROCEDURES
1. Place a small amount of glass wool at the bottom of pasteur pipette.
2. Load the Bio-Gel P-60 into the pasteur pipette.
3. Fill the Bio-Gel P-60 upto 1/2-2/3 height of pasteur pipette.
4.To 50 ul of labelling reaction, add 10 ul of Color Indicator Solution.
5. Mix well using pipettor.
6. Load the labelling reaction mixture at the top of the Bio-Gel.
7.Collect the solution containg labeled probe, where
Blue Dextran
(greenish blue)
coming out.
8. Discard pasteur pipette according to the
radiation safty regulation
.
NOTES
Glass wool is a potential carcinogen. For the safty, work with glass wool in the ventilating hood.
In Bio-Gel P-60 column chromatography, large fragments elute first.
Blue Dextran
behaves as like 300-600 nucleotieds in Bio-Gel P-60 column chromatography.
Unincorporated dNTPs stay with bromo phenol blue.
KIT INFORMATION
REFERENCES
Please send your comment on this protocol to "
editor@MolecularInfo.com
".
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