Removal of Linear Polyacrylamide from DNA by Methanol Precipitation

DNA is commonly analyzed by polyacrylamide gel electrophoresis (PAGE).  DNA fragments of known length can be isolated from polyacrylamide gels by a variety of procedures.

Regardless of the method used to elute DNA from a PAGE gel, some water-soluble linear polyacrylamide usually contaminates the sample.  This is because PAGE gels are crosslinked with approximately 5-10% N,N-methylenebisacrylamide (BIS), however some linear polyacrylamide chains do form without crosslinks, particularly when BIS concentration is low.  Some elution methods solubilize the crosslinked gel by oxidation of the BIS crosslink functional groups, leading to higher concentrations of linear polyacrylamide in the eluted DNA sample.

Linear polyacrylamide is problematic because it has a molecular weight in the same range as PAGE DNA fragments, so it is difficult to attempt gel filtration or dialysis. DNA fragments ~10-1000 bp in length correspond to ~6600-660,000 molecular weight (MW).  It also precipitates in 70% ethanol along with DNA in a common purification step.

Methods 

After PAGE electrophoresis on either a non-denaturing gel a denaturing gel with 7 M urea, electroelution was used to recover 32-P labeled DNA from PAGE gel slices.  Slices were enclosed in a dialysis bag with 1-2 ml of TE buffer and 32-P DNA was transferred from the gel slice to the buffer by immersion in a shallow pool of TAE buffer at pH 7.5.  Approximately 100 V DC was applied and 32-P DNA moved by electrophoresis out of the gel and into the buffer.  Eluted 32-P DNA in the buffer was removed from the bag and the process repeated 2-3 times until the gel slice in the dialysis bag was depleted of 32-P DNA.  Eluates were combined and 32-P DNA was precipitated by addition of 300 mM NaOAc and ethanol to a final concentration of 70%.  After 30 minutes in dry ice samples were centrifuged at 10,000 x g.

Supernatants were decanted and rinsed with cold 70% ethanol, and the 32-P DNA pellet was dried under vacuum.  A visible pellet was always observable, but the nanogram quantities of DNA would be orders of magnitude too small to be visible to the naked eye.

The Question 

So what was the white powder in the pellet?  We were curious, but it did not adversely affect our subsequent steps so we put it on the back burner.

One hypothesis was the powder was talc from our latex gloves, but this was disproven by using powder-free or nitrile gloves.  We still saw white powder in our pellets.

Another hypothesis was that the powder was actually linear polyacrylamide. This observation was made by a young lab technician when it was noticed that pellets from gels with less crosslinker (5% BIS) had more precipitated material than those from gels with more crosslinker (10% BIS).

The later hypothesis was tested by adding ammonium persulfate and TEMED to 10% acrylamide to yield a viscous, but quite liquid, solution of 10% linear polyacrylamide in TAE buffer without any BIS crosslinker.  Dilutions of linear polyacrylamide were spiked with a known amount of 32-P DNA and ethanol precipitated as described above, and at 4 degrees C and room temperature. 

Another dilution series was treated with organic solvents other than ethanol in an attempt to selectively preciptate either the DNA or the linear polyacrylamide to provide a clean DNA sample.

Successful Results

It was found that 50% methanol was effective for precipitating 32-P DNA while leaving linear polyacrylamide in solution.  It is important to keep temperatures above 4 degrees C, otherwise linear polyacrylamide precipitated along with the DNA.