Although additional purification beyond our Gel Filtration (GF) Grade is often unnecessary due to modern RNA, DNA and oligo synthesis techniques, MIDLAND offers five purification options, including:
- Gel Filtration – GF Grade
- HPLC Purification Reversed-Phase – RP Grade
- PAGE Purification (Polyacrylamide Gel Electrophoresis)
- Cartridge Purification
Gel Filtration (GF)
Purification includes MALDI-TOF quality control at no additional charge.
*Minimum of 4 oligos at one time. Length up to 80 bases; typical yield is 1-5 nanomoles.
Our professional staff will be pleased to recommend the purification method that is best suited for your project. Skilled technicians will then perform the purification ensuring the quantity and purity you need.
After the DNA is synthesized and deprotected with ammonium hydroxide, the DNA is purified by a gel filtration column. This purification method removes ammonia, protecting groups, low-molecular-weight molecules, and the shortest DNA sequences. The DNA is quantified by UV spectrophotometry, packaged, and dried. This is the most widely used grade and is suitable for sequencing, amplification, and other applications.
MIDLAND’s HPLC Grade contains these two types of HPLC purification techniques; Anion Exchange (AE) and Reverse Phase (RP). Unless otherwise specified, our experienced staff will determine the HPLC technique that is best for a particular sequence.
DNA or RNA is synthesized as usual, except the hydrophobic dimethoxytrityl (DMT) protecting group is retained at the 5′-end of the sequence upon completion of the last cycle during synthesis. The DNA or RNA is deprotected in ammonium hydroxide or an alternative base and adsorbed to a medium that binds the hydrophobic DMT group. The DMT group is removed with trifluoroacetic acid, and the oligonucleotide is then washed and eluted from the medium with an organic solvent. The product is quantified by UV spectrophotometry, packaged, and dried. This method is suitable for the purification of phosphorothioates and certain other modified oligonucleotides, and for oligonucleotides up to 80 nucleotides in length.
Using this process, DNA or RNA processed through the GF grade is applied to a polyacrylamide gel that has been prepared with the percent of acrylamide best suited to purify the desired, full-length oligonucleotide. The principal band of UV absorbing material, visualized by low-intensity UV shadowing, is cut from the gel and eluted by the crush-and-soak method. The product is precipitated with ethanol, quantified by UV spectrophotometry, packaged, and dried. PAGE Grade is suitable for cloning and is the only method suitable for the purification of very long oligonucleotides. Click here for dual-labeled probes.
Using this process, DNA or RNA is synthesized as usual, except the hydrophobic dimethoxytrityl (DMT) protecting group is retained at the 5′-end upon the completion of the last cycle of synthesis. Deprotection and desalting are done as for GF grade. Cartridge purification is a reverse phase purification method. The cartridge method utilizes small plastic cartridges containing polystyrene copolymer resin as the purification source. The synthesized oligo is pipetted into the cartridge and onto the resin. The oligo of interest is retained on the resin and impurities are washed away with water. A weak protic acid releases the DMT from the oligo, and the purified oligo is then eluted from the cartridge using a solution containing acetonitrile and ammonium acetate. The cartridge purification method is generally the preferred method of purification when a low-cost enrichment of full-length product is wanted, and a substantial reduction in yield can be tolerated. The amount of purified product is generally limited by the capacity of the cartridge. Cartridge purification is therefore only available for synthesis scales of 0.2 micromoles or smaller.