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| Author: Roberto Salvi Source: Contributed by Roberto Salvi |
| Date Added: Tue May 14 2002 |
| Date Modified: Thu Apr 29 2004 |
| Abstract: Glycogen can conveniently substitute for tRNA as a carrier for nucleic acid precipitation. Although Molecular Biology grade glycogen can be purchased from a number of vendors, the main disadvantage is that it is very expensive (e.g., About 100 dollars/20-40 mg). Here we present a simple and inexpensive protocol to prepare a large amount of glycogen which is suitable for any kind of Molecular Biology application (we routinely use it for RNA work). |
Overview
Glycogen can conveniently substitute for tRNA as a carrier for nucleic acid precipitation. Although Molecular Biology grade glycogen can be purchased from a number of vendors, the main disadvantage is that it is very expensive (e.g., About 100 dollars/20-40 mg).
Here we present a simple and inexpensive protocol to prepare a large amount of glycogen which is suitable for any kind of Molecular Biology application (we routinely use it for RNA work).
Glycogen can conveniently substitute for tRNA as a carrier for nucleic acid precipitation. Although Molecular Biology grade glycogen can be purchased from a number of vendors, the main disadvantage is that it is very expensive (e.g., About 100 dollars/20-40 mg).
Here we present a simple and inexpensive protocol to prepare a large amount of glycogen which is suitable for any kind of Molecular Biology application (we routinely use it for RNA work).
Procedure
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. Collect the precipitate by centrifugation and air dry completely the pellet. Weigh the powder so obtained and resuspend it in autoclaved milli-Q water at a convenient stock concentration (10-20 mg/ml is suggested). Store in the freezer in 1-mL aliquots.
PROCESURE
1.0.5g 글리코겐을 3ml의 DEPC DW로 녹인다. (엄청 안녹는다-votexing을 10분이상)
2.phenol 3ml을 넣고 2분 incubation하고 chloroform을 3ml 넣는다.
3. 12000xg 에서 15분 centrifuge하고, 상층액 약 3ml을 딴다. 그리고 e-tube 6개에 500ul의 supernant 넣고 100% merk EtOH을 넣어준다. (완전히 진뜩해짐)
4. 다시 12000xg에 15분 centrifuge 돌려서 pellet을 만든다.
5.pellet을 완전히 말린다.
6.DEPC DW로 녹인다. (엄청 안녹고, 10ul tip으로 pellet을 긁어서 때내고, votexing을 심하게 해주면 녹음.)
동결 건조로 O/N 하고 무게를 잰 후 물에 녹인다.
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. Collect the precipitate by centrifugation and air dry completely the pellet. Weigh the powder so obtained and resuspend it in autoclaved milli-Q water at a convenient stock concentration (10-20 mg/ml is suggested). Store in the freezer in 1-mL aliquots.
2.phenol 3ml을 넣고 2분 incubation하고 chloroform을 3ml 넣는다.
3. 12000xg 에서 15분 centrifuge하고, 상층액 약 3ml을 딴다. 그리고 e-tube 6개에 500ul의 supernant 넣고 100% merk EtOH을 넣어준다. (완전히 진뜩해짐)
4. 다시 12000xg에 15분 centrifuge 돌려서 pellet을 만든다.
5.pellet을 완전히 말린다.
6.DEPC DW로 녹인다. (엄청 안녹고, 10ul tip으로 pellet을 긁어서 때내고, votexing을 심하게 해주면 녹음.)
Sonication이용.
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