Complex carbohydrates do not absorb in the commonly used range of UV (260nm etc) for the most part but they can facilitate precipitation of nucleic acids in the presence of salt and alcohol. This is why glycogen helps recover small quantities of nucleic acid during precipitation. Following the recovery, dissolution in aqueous medium tends to be never a problem.

Chitin is not a positively charged molecule and  it is unlikely to "hold" the DNA through electrostatic interactions. But perhaps it is worth looking into enzymatically degrading complex carbohydrates. Bear in mind that all your additions should be nuclease free. My guess is that either you are starting with very little material and losing it due to incomplete precipitation at low concentration or your system isn't quite nuclease free.

Best, 

Probably, since you are not using phenol, your contaminants are salt and polysaccharides (I don't know if cicada have a high polysaccharides components). Are you washing your DNA with ethanol 70%? You could try to reprecipitate your DNA and increase the washing steps or maybe use DNA columns to better purify it.

Salts you listed do not absorb at 230nm. Looking at your spectral profiles, it seems that you are not extracting much of DNA or RNA. If you were, you should have a distinct peak at ~260nm.

Make sure that your cell lysis is complete and your method provides adequate protection for RNA. Are you using high concentrations of chaotropic reagents such as Guanidinium isothiocyanate at the lysis step? 

Hi Hoa,

I agree with Andre about column purification, but I think in addition to your 260/230 ratio, your 260/280 ratio has pretty bad protein contamination (90-95% protein) and very low nucleic acid yield.  

I would consider rethinking your extraction method? 

Best,

Dave

Is there a reason you are not doing a pheno:chlorophorm extraction?

Complete cell lysis is 1st step to get good DNA. Hope you take care of that.. You can use DNAzole or some such reagent to get good DNA.

Thank you all for your comments.

Actually, I did try various kits (Qiagen DNeasy Blood and Tissue Kit, Labopass Tissue Kit, Accuprep Genomic DNA Extraction Kit) and ethanol precipitation methods (Ammonium Acetate, NaCl 6M) with phenol/chloroform extraction, however, the results were low ratios of purity (A260/280 from 1.1-1.4 and A260/230 below 0.4). 

The main chemical compositions of cicada exoskeleton are chitin (a polysaccharide polymer of N-acetyl-glucosamine as you suggested Andre), cuticular proteins, resilin, and cuticular lipids. Therefore, I think high amount of polysaccharide left-over might be one of the problems. 

I am considering increasing the amount of Proteinase-K in homogenization. Orgininally, for 25mg of exoskeleton,  I put 30ul of 20mg/ml Proteinase-K in manual extraction protocols, and 20ul in kit protocols.

Have you tried CTBT? It is used for M.Tb. extraction.

Start with flash-freezing the insects/bugs in liquid nitrogen, pre-chill a mortar and pestle in LN2, and powder the frozen bugs. The powder can be stored in pre-chilled tubes either in LN2 or at -80ºC until you are ready to extract the RNA or DNA.

Start with this powdered material to extract the RNA/DNA. The best and safest reagent mixtures include Guanidinium isothiocyanate with phenol - followed by the usual...

Have fun! 

I would agree with Tausif,

1) Proper lysis of cell without disrupting the nucleic acid is important. Freezing with Liquid nitrogen and starting is a very good method. And if their is some enzymes to lyse your bug cell wall it will give you good results. Normally for bacterial cell we use lysozyme, i guess u can use chitinase from sigma. It  breaks N-acetyl-glucosamine linkage.

I did use liquid nitrogen for cell lysis, actually.

Thanks for your suggestion, Nithin, I would think more about Chitinase.

Complex carbohydrates do not absorb in the commonly used range of UV (260nm etc) for the most part but they can facilitate precipitation of nucleic acids in the presence of salt and alcohol. This is why glycogen helps recover small quantities of nucleic acid during precipitation. Following the recovery, dissolution in aqueous medium tends to be never a problem.

Chitin is not a positively charged molecule and  it is unlikely to "hold" the DNA through electrostatic interactions. But perhaps it is worth looking into enzymatically degrading complex carbohydrates. Bear in mind that all your additions should be nuclease free. My guess is that either you are starting with very little material and losing it due to incomplete precipitation at low concentration or your system isn't quite nuclease free.

Best, 

You might try a phenol/chloroform extraction of a sample or 3 and re-try PCR.

Thank you so much for your detailed explanation and suggestions David! Your suggestion is exactly what I got from another expert in spectrophotometer. And I learned so much more from your explanation.

I think I have not properly explained the whole DNA extraction protocol I conducted so that it caused my result unclear to you.

Firstly, I am trying to extract DNA from cicada exoskeleton, not cicada tissue. I have extracted DNA from cicada tissue and it worked fine for PCR without any troubles.

Secondly, I used both kits (Qiagen DNeasy Blood and Tissue Kits, Accuprep Genomic DNA Extraction Kit, and Labopass Tissue Kit) as well as not-kit protocols (salting out protocols combined with phenol/chloroform extraction) for DNA extraction and I followed their manual strictly starting from homogenization to lysis, washing steps, and resuspension of DNA. Those protocol worked fine with tissue samples since DNA after extraction yielded good PCR results. However, what I received from exoskeleton was usually high level of contamination (A260/280 ratio of 1.1~1.4 and A260/230 below 0.4).

Thirdly, after DNA extraction, I ran gel electrophoresis to check if there was DNA in my samples before using spectrophotometer to measure DNA concentration and to check for potential contaminants. I got clear bands in the gel, so that I think there was DNA in my samples. Later on, I ran PCR with those DNA samples plus one positive control using DNA from tissue. I got clear and bright band for positive control, but no band for any DNA samples extracted from exoskeleton. 

I hope that my explanation is clear enough for you to understand my situation. Two attached papers showed that exoskeleton does contain DNA and it could be used for genetic study (one as you suggested, and the other used Qiagen Blood and Tissue Kit). Therefore, I suspect that the main problem is contaminants that not totally eliminated. As I said earlier, the main chemical compositions of cicada exoskeleton are chitin (a polysaccharide polymer of N-acetyl-glucosamine), cuticular proteins, resilin, and cuticular lipids. Using proteinase-K helped removing proteins, but may not wash out all chitin. 

That's what I could think of. Please feel free to correct me if I am wrong.

Thank you so so much for your explanation and suggestion David. I will definitely try your two suggestions right away and let you know the results. 

According to your explanation, I think you agree with me on NAG as contaminants in DNA samples. I am considering using chitinase as removal of NAG as proteinase-K as removal of protein. How would you think of that?

David,

I do not wish to interfere but it seems that you may well be making things needlessly complicated for the original poster.

Chitin does not dissolve in water, N-acetyl glucosamine is uncharged*, readily soluble in water (irrespective of what you may have read), and can be easily separated from DNA by most methods of DNA purification whether they are based on ion exchange (like Qiagen's mini preparation kits) or surface adsorption (silica or glass - and, you are correct, most people do not powder their own glass anymore - if one insists on using this method in a home-made column, the powder is available from Sigma - all one needs is to give it a few acid washes  ;) ).

All Hoa needs is larger amount of starting material, assuming there is adequate amount of DNA left in exoskeleton.

______________________

* Resonance and partial +ive charge on N and partial -ive charge on O in a small molecule with net charge 0 at all times makes for a situation where a macromolecule like DNA is not going to be able to interact with N-acetyl glucosamine in a meaningful way that is of any consequence.

Dear Tausif, I did try with double amount of exoskeleton. Previously, I did with the head of the exoskeleton, now I did with both the head and the thorax at the same time, but no better result.

Dear David, I did the purification as you suggested, but the result stayed the same as previously. For the first method which used ammonium acetate, I did not observe any precipitation in the solution. For the second method of TE usage, I did see the white precipitation in the solution. I kept both fractions as you suggested and compared the before- and after-purification, however, they were similar in purify ratios. 

I asked a friend of mine to measure which kind of contaminants could be in the samples using raman spectroscopy measurement. According to the result (attached files), he suggested there might be too much proteins left in the sample, especially those belong to Amide III group.