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Phenol Extraction
One of the most common molecular biology lab procedures,
phenol extraction removes proteins from nucleic acid samples during
isolation, and purifies nucleic acids after enzymatic reactions;
e.g., removal of restriction enzymes during preparation of transcription
template.
Do
Add an equal volume of a 1:1 phenol: chloroform
solution to the aqueous sample and vortex for 1-2 minutes (longer
for larger volumes) creating an emulsion. Please note that isolating
genomic DNA requires gentle mixing because the DNA can be sheared
by vortexing in phenol solutions. Centrifuge at high speed for
2 minutes (10 minutes for larger volumes). Carefully remove the
aqueous or top layer to a new tube, avoiding any flocculent material
at the interface. Even though it is sometimes present in the
aqueous layer, this flocculent material contains proteins and
other debris which should be avoided.
For optimum recovery, the spent phenol
can be back extracted to obtain residual aqueous material left
at the interface. Back extraction involves adding an equal volume
of aqueous buffer to the spent phenol solution. The solution
is vortexed, centrifuged and the aqueous layer removed again
as described above. The two aqueous solutions are then pooled
and precipitated to concentrate the nucleic acids.
Don't
Don't simply invert the extraction a few
times, because inadequate mixing results in insufficient removal
of contaminating proteins. (Inversion is only an efficient extraction
means if it is done over a period of several hours, as with genomic
DNA.)
Don't be tempted to completely remove
the aqueous layer. The purpose of the extraction is to remove
the contaminating proteins, lipids and carbohydrates some of
which can be in the aqueous layer near the interface.
If working with genomic DNA samples, don't
vortex. The samples should be gently and constantly inverted
for several hours to ensure good mixing while preserving the
integrity of the DNA strands.
Tip
To avoid interface material, set your
pipettor to only remove 80-90% of the aqueous phase in the initial
extraction. Use a back extraction to dilute and recover the remaining
10-20% of the aqueous phase.
When performing phenol extractions on large
volumes (10 ml or greater), a short incubation on ice (5 minutes)
can help compact the residue at the interface.
Ethanol Wash
Ethanol washes are performed after salt/EtOH precipitations
to remove any residual salt from the nucleic acid pellet. The wash
employs 70-80% EtOH which will solubilize salts but not nucleic acids.
Do
Add 70 - 80% EtOH to the nucleic acid
pellet. The volume should be sufficient to at least cover the
pellet and wet the sides of the tube when vortexed (there is
no volume too large). Vortex the sample for 1 minute; the pellet
should come loose from the tube and be broken up in the EtOH.
Centrifuge the sample 10 - 30 minutes, to recollect the pellet.
Aspirate off the EtOH.
Don't
Don't just add the EtOH and immediately
decant. The pellet should be vortexed so that the EtOH can penetrate
the sample and solubilize salt.
Don't forget to respin! The pellet must
be firmly reaffixed to the tube so that it is not lost during
aspiration.
Double Aspiration
Double aspiration is useful for removing the last
traces of EtOH supernatant after precipitations. It involves a second
quick spin and aspiration to ensure removal of any precipitation
supernatant e.g. on the walls of the tube, that might interfere with
downstream steps of the protocol. We recommend it in Ambion's RPA
IIé protocol, and for RNA probe template preparation.
Do
After pelleting the precipitation, aspirate
the precipitation supernatant off the nucleic acid pellet. Follow
immediately with a quick 1-2 second centrifugation and aspirate
again.
Aspiration can be done with a syringe
needle or a drawn-out Pasteur pipette connected to a vacuum source
with a trap. Alternatively, a drawn-out Pasteur pipette can be
used with a pipette bulb. To make drawn-out Pasteur pipettes,
soften the pipette tip with a flame and draw the tip out with
forceps, break the tip at the narrowest point and flame polish
if needed.
Slant the tube, pellet facing up, and
aspirate from beneath.
Don't
Don't let the tubes sit very long between
aspirations as the pellet may come loose.
Resolubilizing RNA Pellets
During RNA isolations and purifications, it is
often necessary to precipitate the sample. Resolubilizing the RNA
pellet after precipitation can be time-consuming and the presence
of proteins or other contaminants can make it difficult.
Do
After precipitation, perform double aspiration
as above and air dry for 5-10 minutes.
Use the largest volume of solute possible
to increase solubility. At Ambion, we believe the less dry the
pellet, the easier it is to solubilize.
Don't
Don't dry by vacuum centrifuge (speed
vac).
Tip
To aid resolubilization of overdried RNA,
store the RNA pellet and solute together overnight at -80°C.
The freeze-thaw process helps in subsequent solubilization.
Reprinted from Ambion's TechNotes
Newsletter 4:4, © 1998
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