Dear all,
I am extracting DNA from cicada exuviae and the products are contaminated with protein. I have tried different kits (DNeasy from Qiagen, Accuprep from Bioneer, and Labopass from Cosmo) and also I tried with ethanol precipitation method but none of them showed pure DNA products. Since the DNA products are contaminated, I could not get any PCR products from them. I tried to precipitate DNA using ethanol 100% and NaCl 5M but unsuccessfully.
Can you help suggesting other options that I can try? Thank you!
Try a proteinase K digest at 55°C over night and and subsequently a phenol chloroform extraction
Hello. Precipitation is mont efficient for removing protein. You have to perform a phenolchlorophorm extraction for removing them.
But before have you checked your DNA, in order to verify that teir are not dgradated? you can depose 500ng by well on a 0,8% aggarose gel and let them migrated at 125V for 25mn. If you DNA are ok you can performed a phenol chlorophorm purification.
For the precipitation you need to have at least 100µL of DNA if it not the cases dilute your sample. Add volume to volume 25:24:1 phenol/chloroform/isoamyl alcohol. Vortex vigorously for 10 sec and centrifuge 15mn at 10000g. (For this step you can use phaselock gel tube that permit to isolate the aqueous phase from the phenolic phase). Carefully remove the top (aqueous) phase containing the DNA using a 200-μl pipettor and transfer to a new tube. If a white precipitate is present at the aqueous/organic interface, repeat this steps. After proceed to the precipitation with 1/10 volume of 3 M sodium acetate, pH 5.2 and 2.5 volume of cold ethanol 100%. mix by inverting the tube. Put the tube at least 1h at -20°C or 15mn at -80°C. Centrifuge 30mn at 10000g. remove the supenatent and wash the pellet with 1 volume of EtOH 70%. Centrifuge 5mn at 10000g, remove the suparnatent and air dry the pellet. When it's dry resuspent in TE pH=8.
Hi there,
I don't think the contamination issue is the actual reason why you don't get your PCR experiment to work (unless DNAse and proteases are among the contaminants). If your DNA sample is only meant to be used as a Template for PCR, I would suggest you to focus on PCR protocol optimization too. In my experience, I have run a lot of PCR for screening using very crude genomic DNA preps or even whole cells as templates.
In the past I've had problems with RE digestion of plant genomic DNA rich in polyphenols. Getting rid of these compounds was very important to get good RE digestions.
You are extracting DNA from insects, and hence getting rid of chitin and some oxidized phenolic compounds from insects (that may perhaps be interfering with PCR amplifications) may be helpful in getting clean DNA. Here is the protocol that may help you.
http://www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/v12n2-4/709
Good luck
Thank you all for your suggestion! I will try with Christelle Tesson first and let you know if it works.
I have tried running PCR with those DNA samples but I got either no results or only smears. That's why I think about purifying my samples.
I have read Oliveira et al. 2009 but not tried two protocols proposed in there yet. I want to try both of them later.
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