Gel filtration and spun column chromatography

목차

1. Gel - filtration chromatography
2. Spun-column chromatography

본문내용

This technique, which employs gel filtration to separate high-molecular weight DNA from smaller molecule, is used most often to separate unincorporated labeled dNTPs from DNA that has been radiolabeled. however, it is also used at several stages during the synthesis of double-stranded cDNA, during addition of linkers to blunt-ended DNA, to remove oligonucleotide primers from polymerase chain reaction, and , in general, whenever it is necessary to change the composition of the buffer in which DNA is dissolved.

Two methods are available : conventional column chromatography, which is used when it is necessary to collect fractions that contain components of different sizes, and centrifugation through gel matrices packed in disposable syringes, which is rapid method used to free DNA from small molecules. The two most commonly used gel matrices are Sephadex and Bio-Gel, both of which are available in several porosites. Sephadex G-50 and bio-Gel P-60 are ideal for purifying DNA larger than 80 nucleotides in length. Smaller molecules are retained in the pores of the gel, whereas the larger DNA is excluded and passes directly through the column. Bio-Gel P-2 can be used to separate oligonucleotides from phosphate ions or dNTPs. Bil-Gel is supplied in the form of a gel and need only be equilibrated in running buffer before use. Sephadex is supplied as a powder that must be hydrated before use.

참고 자료

Molecular cloning - A Laboratory Manual , 3rd Edition, Joseph Sambrook
단백질 실험노트 - 김승호 외 1명, 2001년, 월드사이언스, p 145-155
Biochemistry - 5th Edition, Jeremy M. Berg, p 81
Amersham web site