9 replies to this topic

[b*g]

While performing a DNA extraction using the Qiagen midi prep kit, I inadvertently poured the isopropanol through the column.  Does anyone know if this will create a problem?

[perneseblue]

Where are you in the protocol? Has the DNA been eluted from the column?

[b*g]

perneseblue, on 19 December 2010 - 06:00 PM, said:

Where are you in the protocol? Has the DNA been eluted from the column?

Yes, I eluted the DNA (after washing the column with 20ml QC buffer), then added the isopropanol, also through the column, instead of directly to the eluate.  I am hoping there was nothing left on the column that would have contaminated the DNA.

[phage434]

This is unlikely to cause any problems.  Likely, any contaminants in the column have already eluted with the PE ethanol washes.  You may (almost certainly did) lose some isopropanol in the column, and may need to add more to the DNA sample to precipitate it.  The amount is not critical, as long as there is sufficient.

[b*g]

phage434, on 20 December 2010 - 05:08 AM, said:

This is unlikely to cause any problems.  Likely, any contaminants in the column have already eluted with the PE ethanol washes.  You may (almost certainly did) lose some isopropanol in the column, and may need to add more to the DNA sample to precipitate it.  The amount is not critical, as long as there is sufficient.

Hi - Yes, I thought it wouldn't be a problem, too.  However, after nanodropping the samples today, I can say "Don't do what I did!".  The DNA was completely degraded.  My thought is that perhaps the isopropanol pulled some column material into the sample.  I don't know what the columns are made of, but clearly something came off that destroyed the DNA.

[phage434]

I'm confused.  As far as I know, one cannot determine if DNA is degraded by using a nanodrop.  Exactly what did you measure to conclude this?

[b*g]

phage434, on 20 December 2010 - 06:27 PM, said:

I'm confused.  As far as I know, one cannot determine if DNA is degraded by using a nanodrop.  Exactly what did you measure to conclude this?

The nanodrop indicated extremely low 260:280 (below 1) values and low (sometimes negative) 260:230 values. The graphs, instead of being nice curves (as is usual with good DNA or RNA) were just spiky, like static. The concentrations were in the 50-300ng/ul range, so I tried a 1:10 dilution, thinking my samples might be too concentrated, and while this brought the concentrations down (as expected), the graphs still looked like static and the ratios were still very, very poor.  I concluded from this that any DNA present in the samples was degraded and/or contaminated.  But it is true, to get a complete picture of the state of the nucleic acids present, one would want to Bioanalyze.  I didn't think these samples were worth burning a chip for!

[bob1]

Bioanalyse! - just run a gel!

[perneseblue]

OD ratios do not say anything about the integrity of your DNA. Even degraded DNA, pure degraded DNA will give nice ratios and a good curve.

The odd ratios do indicate that the DNA sample is contaminated by something... what do intend to use this DNA for?

I believe a phenol chloroform extraction should clear up most contaminants.

And yes, do run the DNA on a gel to check for integrity.

[b*g]

OK, everyone! Thanks for all your help!  I'm glad to know about the OD ratios not being a good indication of DNA quality, that's very useful.  Yes, I will run a gel.  After the holidays!