I'm doing DNA extractions from Spisula (surf clam) adductor muscle, using the Qiagen DNEasy kit. For some reason, I'm getting low yields, cloudy DNA, and a HUGE peak at 230nm when I quantify it using our Nanodrop. I'm aware that some reagents also make a peak at 230, but I can use the same kit and the same technique, and this doesn't happen with gill tissue or really any other tissue type I've tried. I also do not usually get cloudy DNA.
We've even tried ethanol precipitations, and whatever this is coprecipitates there as well. Has anyone experienced this? I really need to know what it is, to know if it will interfere with our downstream NGS applications.
- I am not sure of the contaminating principle in your particular extractions, but if ethanol ppt alone does not remove the contaminating principle, then a second column purification or better still column purification followed by Trizol and chloroform plus ethanol ppt followed by 2 x room temp 70% ethanol washes might
- Better still you could just do the Trizol _ chloroform- Isopropanol-70% wash x 2 and that could work the best and not compromise your yields too much
- Or Trizol-Chloroform then column purification
- I don't know the composition of your starting material but 'exotic' muco polysaccharides, sialoproteins etc can be a problem for columns to remove and can also block these traditional silica based columns
- In contrast organic extractions are more robust for these atypical materials
- Thus, one advantage of performing a preceding Organic phase extraction is that you remove these substances that interfere with good column extraction, allowing the column to do well what it is mean to do, i.e. remove salts and residual protein from your nucleic acid
Dear Harmoney, our experience using high salt ppt of lysis digests followed by ppt of DNA from the supernatant with alcohol is that glycogen also co-precipitates. This can form a cloudy solution in shellfish. There can be a lot in muscle and for shellfish muscle it can be ~25% dry weight glycogen. This usually does not interfere with downstream reactions if its not excessive. Some kits reduce this problem but it will still be present. Summary as Laurence has suggested you have to do a two stage purification system and consider "tweaking" conditions.
John Colin McEwan , does glycogen make a peak at 230nm for spectrophotometry? Because it sounds like that explains our cloudiness, but not necessarily the only issue happening.
Harmoney I am not sure about a peak at 230nm for glycogen but I do know the ratios are affected. There could be other polysaccharide linked contaminants as well. Basically high molecular weight compounds that have low solubility in alcohol. We do GBS and use Zymo tissue extraction kits for shellfish. We have the same issues but it works for our downstream reactions. However, it may not work for many other systems.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts