EDTA, guanidine salts and oligosaccharides can all absorb around the 230 wavelength. The PE wash step is used to remove the left over gel and the salts from the column.
EDTA is usually not a component of wash buffers. But since the buffer formulations in these kits are proprietary, there is no way to know if a low level of EDTA is contained in PE. EDTA is a nuclease inhibitor so adding it into buffers may be a way of ensuring DNase activity is eliminated.
When mistakes occur, it can be hard to troubleshoot when you don’t know the formulas for the solutions you are using.
But the chemistry is really simple (bind silica with chaotropic salts, wash away salts with ethanol, dry to remove the ethanol, elute in low salt buffer or water) and fixing slip ups does not have to mean starting over.
So what could have happened to your sample?
Since you eluted too early in the protocol, your sample would have contained guanidine salts, agarose and prehaps EDTA.
When you dried it down, you also concentrated those chemicals. With a 70% ethanol wash to remove residual salt, you may have removed some of it, but probably not all of it because when guanidine solidifies, it becomes difficult to resolubilize and usually needs some heating. So you did get rid of the ethanol, but probably not the guanidine.
If the contaminants were simply salts or EDTA, then ethanol precipitation would be a better way to clean up the DNA instead of drying it down. If the contaminant was gel residue, ammonium acetate should be used for the precipitation salt because it will keep oligosaccharides soluble.
Forget the chemistry – just use a kit
If all of that chemistry is sounding like a bit too much hard work, you will be relieved to know that there is another solution that you could try if this happens again:
Simply re-bind the DNA to a new gel column by adding back the QG binding buffer or if you have a PCR clean up kit in your lab, you could use that to clean up the DNA on a new column. You should be able to recover 80% back (according to manufacturer data).
More gel extraction help…
You can get more tips on gel extraction (15 in total!) in a couple of articles, one bymyself and one by Nick.
But now that I am in gel extraction troubleshooting mode… here are a few more:
1. Always perform the additional QG wash to remove residual gel debris, especially if you pushed the limit of the max allowed for gel size, and then perform a second ethanol (PE) wash to make sure all the salt is removed.
You can perform a second wash with 80% ethanol instead of the PE wash so you don’t run out. Of course, dry the column before elution, but at least if you do forget, then the speed vac method will work to clean it up.
2. Only 100% anhydrous ethanol should be used in silica column wash buffers. Using denatured alcohol (typically the outer container will say 95% alcohol) will cause many problems with the drying step.
Denatured alcohol contains chemicals such as isopropanol or benzene that do not evaporate quickly and this carries over into the DNA.
You know when you used the wrong ethanol when your DNA smells funny or it won’t freeze in the -20C. And when you try and load it on a gel, it floats, even in loading dye.
If you are using a Nanodrop to quantify the DNA, the readings tend to not accurately reflect the purity if the sample is near the lower level of detection for the instrument. I usually see this effect at around 10ng/ul and below.
When the DNA concentration is very low, the 260 reading is not peaking so the scan looks more like a straight line instead of a bell curve. But if the scan looks like a ski slope (starting out high at the 220-230 side and then sloping down as it gets to 260), then you do have some salt or other contamination in there. The scan will tell you more information, so the 260/230 may be fine and it may just be an effect of low yield.
If you are using a spectrophotometer, the same effect will apply. And, of course, for UV analysis of DNA, if you eluted the DNA in a buffer with EDTA, such as TE, then blank the instrument in TE.
You’ve tried all the usual stuff, and checked the primer sequences twice, but still can’t get that PCR fragment amplified. It’s time to enter the strange world of PCR additives. Over the years a variety of additives have been shown to enhance PCR reactions in certain situations. Here is a summary of some of the […]
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