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Glycogen

Glycogen, molecular biology grade	#R0561	2x0.25ml
Glycogen, RNA grade	#R0551	2x0.1 ml
Related Documents (in pdf, ~30-50 KB): Certificate of Analysis: #R0561, #R0551 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Optimal for recovery of oligonucleotides (>8 bases) and low amounts of DNA/RNA (>20 pg) from diluted solutions.
• Free of host DNA/RNA.
• Forms clearly visible pellets.
• Does not interfere with enzymatic reactions.
• Does not interfere with gel electrophoresis of nucleic acids.
• Does not interfere with spectrophotometrical determination of DNA/RNA concentration, (A_260-280 measurements).
• Compatible with DNA transformation and in vitro transfection.

Description
Glycogen is a highly purified polysaccharide derived from oysters. It is an inert carrier which significantly increases the recovery of nucleic acids by alcohol precipitation. Nucleic acids are precipitated when their negatively charged phosphate groups are exposed because of depletion of the hydration shell by alcohol. Salt cations, like Na⁺, are attracted to these negatively charged groups. Therefore, they reduce the repulsive forces between the polynucleotide chains to the point where a precipitate can form. The Glycogen is insoluble in ethanolic solution and forms a precipitate that traps the target nucleic acids. During centrifugation, a visible pellet is formed, which greatly facilitates handling of the target nucleic acids. The Glycogen quantitatively precipitates nucleic acids from diluted solutions with a higher efficiency than that of tRNA, linear polyacrylamide or sonicated DNA (1-4).
Fermentas offers two formulations of aqueous Glycogen solutions which are inert co-precipitants of nucleic acids, providing the fast and efficient DNA or RNA recovery.
Glycogen, molecular biology grade (#R0561), recommended for DNA precipitation.
New Glycogen, RNA grade (#R0551) has been specifically designed for RNA precipitation.

Source
Oysters.

Molecular Weight
Glycogen molecules are highly branched structures which are composed of thousands of glucose molecules bonded to each other. The molecular weight of the largest individual glycogen molecule appears to be 8 million. Such a molecule contains about 50,000 glucose molecules.

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases, ribonucleases, phosphatases, proteases and nicking activity is confirmed by appropriate quality tests.
Glycogen, molecular biology grade, is functionally tested for DNA precipitation.
Glycogen, RNA grade, is functionally tested for RNA precipitation.

Storage
Store at -20°C.

Protocol for Nucleic Acid Precipitation from Diluted Solutions

1. Add 1/10 volume of 3 M sodium acetate (or 2 M sodium chloride, or 5 M ammonium acetate) to DNA solution.
2. Add Glycogen to a final 0.05-1 µg/µl concentration. Use up to 1 µl of Glycogen per 20 µl of the solution.
3. Add 1 volume of isopropanol (or 2.5 volumes of ethanol) to the solution. Mix gently but thoroughly. Ethanol is recommended for precipitation of smaller than 200 b/bp RNA/DNA fragments.
4. Incubate the mixture at -20°C for up to 60 min, or at -70°C for 30 min.
5. Centrifuge the mixture for 10-15 min at 10,000 rpm. Discard the supernatant.
6. Rinse the pellet with cold 70% ethanol. Air-dry the pellet.
7. Dissolve DNA in Water, nuclease-free or TE buffer.
8. Dissolve RNA in DEPC-treated Water.

Note

• 5 µg of Glycogen (0.25 µl) forms a visible pellet in a microcentrifuge tube.

• Recommended final Glycogen concentration in a precipitation mixture is 0.05-1 µg/µl.
• For precipitation of oligonucleotides, 1 µg/µl final Glycogen concentration is recommended.
• Longer incubation at lower temperature provides better recovery of nucleic acids.
• Avoid over-drying the pellet, as it then takes more time to dissolve.
• Up to a final concentration of 8 µg/µl, Glycogen does not interfere with most downstream applications: PCR, DNA sequencing, DNA digestion by endonucleases, ligation using T4 DNA Ligase, cDNA synthesis using Reverse Transcriptases (#EP0351, #EP0451, #EP0441),  DNA labeling with T4 Polynucleotide Kinase, random priming with DNA polymerases, in vitro transcription using T7 RNA Polymerase and DNA amplification using phi29 DNA Polymerase.
• Up to 8 µg of Glycogen does not interfere with bacterial transformation.
• Up to a final concentration of 0.4 µg/µl, Glycogen does not affect in vitro transfection of eukaryotic cells with ExGen 500 in vitro Transfection Reagent.

References

1. Tracy, S., Improved rapid methodology for the isolation of nucleic acids from agarose gels, Prep. Biochem., 11, 251-268, 1981.

2. Helms, C., A new method for puryfying lambda DNA from phage lysates, DNA, 4, 39-49, 1985.
3. Hengen, P. N., Methods and reagents - Carriers for precipitating nucleic acids, TIBS, 21, 224-225, 1996.
4. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the Third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, A8.12-8.13, 2001.

Updated balandžio 25, 2008 08:52