What is the purpose of glycogen in the human DNA extraction protocol?
Glycogen is a polymer which is used to trap the nucleic acids (DNA, rRNA,RNA…). In ethanol Glycogen is in soluble so it forms polymer structure and traps the nucleic acids and forms pellet with nucleic acids trapped during the centrifugation…
How much glycogen do I add to RNA?
Add glycogen to a final 0.05-1 μg/μL concentration. Use up to 1 μL of glycogen per 20 μL of the solution. For precipitation of oligonucleotides, do not use higher than 1 μg/μL final glycogen concentration.
Does glycogen inhibit reverse transcriptase?
A concentration of ≤ 2 mg/mL glycogen has no effect on SuperScript® II Reverse Transcriptase (Gerard, 1994). T4 RNA Ligase is not inhibited by samples precipitated in the presence of 0.02 mg/mL glycogen (Apte, 1993). Glycogen can interfere with DNA-protein interactions (Galliard, 1990).
Why do we need to precipitate glycogen from the solution?
Glycogen effectively precipitates nucleic acids in solution to enhance the formation of a visible pellet upon centrifugation.
Is glycogen soluble in ethanol?
Glycogen is insoluble in ethanol solution; in the presence of salts it forms a precipitate that traps the target nucleic acids. The molecular weight of the largest individual glycogen molecule containing about 50,000 glucose molecules appears to be 8 million.
How do you prepare glycogen solution?
Start by dissolving 5 gr. of glycogen (from Oyster, Sigma G 8751) in 10 ml of milli-Q water. Extract once with one volume of Phenol followed by one extraction with one volume of Chlorophorm. Add one volume of absolute ethanol to the supernatant, at this point the glycogen will precipitate instantaneously.
Does glycogen interfere with PCR?
† At a final concentration up to 8 µg/ µL, Glycogen does not interfere with PCR, DNA sequencing, DNA digestion by endonucleases, ligation, cDNA synthesis, DNA labeling, in vitro transcription, or bacterial transformation.
Why glycogen is used in RNA isolation?
Glycogen is a polysaccharide that is insoluble in ethanol and when added to DNA or RNA, will trap the nucleic acids as it precipitates and allow for a more visible pellet. In RNA liver preparations you can actually observe a pellet coming through the column and forming at the bottom of the collection tube.
What is the role of isopropanol in RNA extraction?
While isopropanol is somewhat less efficient at precipitating RNA, isopropanol in the presence of NH 4+ is better than ethanol at keeping free nucleotides in solution, and so separating them from precipitated RNA. RNA precipitation is faster and more complete at higher RNA concentrations.
Is glycogen a structure?
Glycogen is the analogue of starch, a glucose polymer that functions as energy storage in plants. It has a structure similar to amylopectin (a component of starch), but is more extensively branched and compact than starch. Both are white powders in their dry state.
Is glycogen insoluble in cold ethanol?
Glycogen is insoluble in ethanol solution; in the presence of salts it forms a precipitate that traps the target nucleic acids. Glycogen molecules are highly branched structures composed of thousands of glucose molecules bonded to each other.
What happens when glycogen is added to RNA?
Glycogen is a polysaccharide that is insoluble in ethanol and when added to DNA or RNA, will trap the nucleic acids as it precipitates and allow for a more visible pellet.
How to isolate RNA from a cell sample?
Isolation of RNA from small quantities of tissue (1 to 10 mg) or Cell (102 to 104) Samples: Add 800 µl of TRIZOL to the tissue or cells. Following sample lysis, add chloroform and proceed with the phase separation as described in step 2. Prior to precipitating the RNA with isopropyl alcohol, add 5-10 •Ïg RNase-free glycogen (Cat.
Can you use glycogen to co precipitate DNA?
Saad Jan Yes you can use glycogen its quite helpful. It helps to co precipitate DNA and if visibility is an issue due to low quantity of the DNA in your sample, you can use GlcoBlue which imparts a blue color to the pellet during ethanol precipitation. as for the concentration of glycoblue that should be used during the protocol is 50 ug/mL.
How long to incubate isopropyl alcohol for RNA wash?
Use 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at no more than 12,000xg for 10 minutes at 2 to 8°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. 4. RNA WASH.