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Phenol Chloroform Isoamyl Alcohol (25:24:21) DNA purification

Phenol Chloroform DNA extraction DNA purification Ethanol Precipation

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#1 Epigeneticist

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Posted 29 May 2013 - 05:32 PM

First time using phenol chloroform isoamyl alcohol (25:24:1) for DNA purification. Product is purchased and not prepared in the lab. At storage conditions of 4C, the liquid has two phases, upper and lower.

What are the two layers? Which layer to use?

Do I mix and quickly use?

Do I need to let the bottle warm to room temperature before use?

Will this cause the two phases to mix then I can use?

Thanks!

#2 christy

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Posted 29 May 2013 - 05:35 PM

Phenol extraction and Ethanol Precipitation of DNA
This protocol describes the most commonly used method of purifying and concentrating DNA preparations using phenol extraction and ethanol precipitation; it is appropriate for the purification of DNA from small volumes (<0.4ml) at concentrations ≤1mg/ml.
Materials
-DNA to be purified (≤1 mg/ml) in .1 to .4 ml volume
-25:24:1(v/v/v) phenol/chloroform/isoamyl alcohol (made with buffered phenol: Support Protocol 1)
-3M sodium acetate, ph 5.2
-100% ethanol,ice cold
-70% ethanol, room temperature
-Ultra pure water
1. Add an equal volume of phenol/chloroform/isoamyl alcohol to the DNA solution to be purified in a 1.5-ml microcentrifuge tube.
2. Vortex vigorously 10 sec and microcentrifuge 15 sec at room temperature
3. Carefully remove the top (aqueous) phase containing the DNA using a 200 microliter pipettor and transfer to a new tube. If a white precipitate is present at the aqueous/organic interface, reextract the organic phase and pool the aqueous phases.
4. Add 1/10 volume of 3M sodium acetate, pH 5.2, to the solution of DNA. Mix by vortexing briefly or by flicking the tube several times with a finger
5. Add 2 to 2.5 vol (calculated after salt addition) of ice-cold 100% ethanol. Mix by vortexing and place in crushed dry ice for 5 min or longer.
6. Spin 5 min in a fixed-angle microcentrifuge at high speed and remove the supernatant.
7. Add 1ml of room temperature 70% ethanol. Invert the tube several times and microcentrifuge as in step 6.
8. Remove the supernatant. Allow to air dry for 15 minutes
9. Resuspend DNA pellet in 100 microliters of Ultra pure water
Protocol adapted from:
Ausubel, F.; Brent, R.; Kingston, R.; Moore, D.; Seidman, J.G.; Smith, J.;Struhl, K. Short Protocols in Molecular Biology(1995), 3rd ed., Unit 2.1: page 2-3.

#3 Epigeneticist

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Posted 29 May 2013 - 05:45 PM

Phenol extraction and Ethanol Precipitation of DNA
This protocol describes the most commonly used method of purifying and concentrating DNA preparations using phenol extraction and ethanol precipitation; it is appropriate for the purification of DNA from small volumes (<0.4ml) at concentrations ≤1mg/ml.
Materials
-DNA to be purified (≤1 mg/ml) in .1 to .4 ml volume
-25:24:1(v/v/v) phenol/chloroform/isoamyl alcohol (made with buffered phenol: Support Protocol 1)
-3M sodium acetate, ph 5.2
-100% ethanol,ice cold
-70% ethanol, room temperature
-Ultra pure water
1. Add an equal volume of phenol/chloroform/isoamyl alcohol to the DNA solution to be purified in a 1.5-ml microcentrifuge tube.
2. Vortex vigorously 10 sec and microcentrifuge 15 sec at room temperature
3. Carefully remove the top (aqueous) phase containing the DNA using a 200 microliter pipettor and transfer to a new tube. If a white precipitate is present at the aqueous/organic interface, reextract the organic phase and pool the aqueous phases.
4. Add 1/10 volume of 3M sodium acetate, pH 5.2, to the solution of DNA. Mix by vortexing briefly or by flicking the tube several times with a finger
5. Add 2 to 2.5 vol (calculated after salt addition) of ice-cold 100% ethanol. Mix by vortexing and place in crushed dry ice for 5 min or longer.
6. Spin 5 min in a fixed-angle microcentrifuge at high speed and remove the supernatant.
7. Add 1ml of room temperature 70% ethanol. Invert the tube several times and microcentrifuge as in step 6.
8. Remove the supernatant. Allow to air dry for 15 minutes
9. Resuspend DNA pellet in 100 microliters of Ultra pure water
Protocol adapted from:
Ausubel, F.; Brent, R.; Kingston, R.; Moore, D.; Seidman, J.G.; Smith, J.;Struhl, K. Short Protocols in Molecular Biology(1995), 3rd ed., Unit 2.1: page 2-3.


I actually have this protocol. However, my question is related to the phenol chloroform isoamyl alcohol reagent itself. At 4C, the solution is two layers.

Do I need to let the bottle come to room temp before use and allow layers to mix?

Or do I need to use the lower or upper phase of the reagent?

#4 phage434

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Posted 29 May 2013 - 06:25 PM

Use the lower phase, which is the phenol/chloroform. The upper phase is water + buffer, holding the pH of the reagent at the correct value. Don't use plastic serological pipets, which will dissolve in chloroform. Polypropylene or glass is fine. You don't need to heat it to RT.
Read further, especially about safety, here:
http://openwetware.org/wiki/Phenol





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