Hi Chandra,

what you have there is a blast from the past. That was pretty much a standard protocol for extraction of DNA from agarose before the arrival of kits. It works fine, I used it many times in the 80's.

A few of points:

Melting the agarose at 65-70oC in the H2O improves extraction.

It's best if the phenol is Hot (65-70oC) as well.

You will get a white gunk layer between the two phases (the agarose?) after centrifugation, don't take this.

Phenol is dangerours, check your labs procedures before using it. 

-Richard

 Dear Chandhra Lekha,

 Phenol is a useful compound for breaking down superfluous cell materials that would otherwise contaminate the nucleic acid sample. There are two reasons why phenol makes such an effective purifier. The first is that it is a non-polar compound. Because nucleic acids are highly polar, they do not dissolve in the presence of phenol. The second is that phenol has a density of 1.07 g/cm3, which is higher than the density of water (1.00 g/cm3). Thus, when phenol is added to a cell sample solution the water and phenol remain separate. Two “phases” form when phenol is added to the solution and centrifuged. There is an aqueous, polar phase at the top of the solution containing nucleic acids and water, and an organic phase containing denatured proteins and other cell components at the bottom of the solution. The aqueous phase is always on top of the organic because, as mentioned above, phenol is denser than water. Nucleic acids are polar, and therefore stay in the aqueous phase, whereas non-polar cellular components move into the organic phase. After phenol has been added to the sample it is centrifuged and the aqueous and organic (“Phenol”) phases form.

Think this information useful.

Sreedhar

Hi Chandra,

what you have there is a blast from the past. That was pretty much a standard protocol for extraction of DNA from agarose before the arrival of kits. It works fine, I used it many times in the 80's.

A few of points:

Melting the agarose at 65-70oC in the H2O improves extraction.

It's best if the phenol is Hot (65-70oC) as well.

You will get a white gunk layer between the two phases (the agarose?) after centrifugation, don't take this.

Phenol is dangerours, check your labs procedures before using it. 

-Richard

Dear Chandra

i agree with Richard. I did this routinely in the late 80s and early 90s. An alternative to narration is spinning your gel slice through a glass wool column which separates agarose gel from aqueous DNA before performing phenol chloroform extraction and ethanol precipitation as below: 

1.  Make sure the phenol is tris saturated by shaking vigorously and leaving for at least 1 hour before taking the bottom lower phase.  Vortex your phenol with macerated gel then spin for 5 min to obtain good phase separation.  Do not reduce the length of thus spin as this can lead to incomplete phase separation and contamination with interface material when you remove upper aqueous phase 

Make sure your phenol is less then 1 month old if kept in fridge or hot older phenol store in freezer and thaw plus shake to tris saturate before use.  Older phenol kept at 4c to room temp oxidises and oxidised phenol degraded DNA !!!

2.  Having extracted with phenol then extract with an equal volume of chloroform_isoamylalcohol (IAA) (24:1):  this removes the phenol which can degrade your nucleic acid after prolonged contact ( and also lipids and residual protein).  Vortex and spin for 5 min as before

3.  Most importantly whether you perform just a phenol or phenol + chloroform extraction precipitate your DNA to concentrate and remove impuritities: 

Do this by adding 3 volumes of ethanol + 1/10 volume 3M sodium acetate pH 4-5.  

Incubate for 15 min to 1 hour at -20C

Spin at 13K for 15 min in an eppendorf to ppt DNA

Remove most of the supernatant

add back 500ul 70% ethanol

vortex briefly then respin for 5 min to re pelley

Remove most of supernatant 

Spin for 1 min to bring down residual liquid on sides of tube

remove liquid with a p10 tip then air dry pellet for 15 min at room temp

Nowadays...get an already used spin column and its 2.0ml collection tube. You can remove the glass filter/fleece and keep the ring and solid support membrane.

Insert the membrane support and ring together and add 500ul of 70% EtOH to wash the membrance. Centrifuge with the collection tube and remove the ethanol.

Remove the ring and add 2-3 of sterile filter papers. These filter papers can be prepared by using punchers which makes the size of the filter paper just nice to be inserted into the column. Insert the ring and you get a modern version of gel isolation using the "freeze & squeeze method".

You can precipitate the collected supernatant using Laurence's step.

Chandra, 

It is very important that you usevbuffer saturated phenol with a pH between 7.8-8.0. At lower pH your DNA may end in solution in the organic phase. 

From what you have mentioned. it appears that agarose is not getting dissolved in Phenol. Instead, the DNA from the band is getting extracted in the non-aqueous layer of Phenol chloroform and you can precipitate it with cold ethanol (A routine DNA extraction procedure).