I am trying to extract DNA from naturally molted feathers with standard phenol:chloroform:iaa extraction. I first lyse the samples in lysis buffer (50 mM Tris-HCl, pH 8, 20 mM ethylenediaminetetraacetic acid [EDTA], pH 8, 2% sodium dodecyl sulfate) with 5 µl of 20 mg/ml proteinase K solution. Than I proceed with phenol:chloroform extraction followed by ethanol precipitation. I couldn't get any results as of yet. I can't seem to find the problematic step but my samples don't completely dissolve in lysis buffer. They don't even undergo a change. What could be the reason that I can't lyse the samples?
Lysozyme digests peptidoglycans and chitodextrins from bacterial cell walls, and will not do anything with your feather material.
Feathers are made of beta-Keratin. Your extraction buffer cannot dissolve the actual feather material. The easiest would be to get a fresh feather that includes the base with residual skin cells and/or blood. It's known to be much more difficult to get DNA from molted feathers. With fresh feathers you never have to worry about digesting the keratin, since the cells and blood on the surface will readily dissolve in your lysis buffer and you will get great DNA, with the feather material still intact.
What do you mean you don't get any results? You don't get enough DNA to see on gel or measure spectroPhotometrically? That's not important, even if you can't see or measure it, just use it in a PCR and see what you can amplify. If your PCR didn't work, yes you have to improve your extraction method.
If the molted feathers is the best material you have, need a better DNA isolation method. You can do a pretty good job digesting the keratin by using DTT that slowly breaks the SS crosslinks between the keratin monomers, so you can get to the additional (low amount of) DNA present inside the feather (with prolonged incubation at elevated temp). The finer you can cut/grind your feather material, the faster the dissolving will be.
Check this paper, it has a protocol for DNA isolation from old and molted feathers:
http://rspb.royalsocietypublishing.org/content/276/1672/3395.full
Goodluck
Hi,
Try using lysozyme, better yet check Pressure Bisciences' technology:
http://www.pressurebiosciences.com/pub/media.htm
Best,
Klara
Lysozyme digests peptidoglycans and chitodextrins from bacterial cell walls, and will not do anything with your feather material.
Feathers are made of beta-Keratin. Your extraction buffer cannot dissolve the actual feather material. The easiest would be to get a fresh feather that includes the base with residual skin cells and/or blood. It's known to be much more difficult to get DNA from molted feathers. With fresh feathers you never have to worry about digesting the keratin, since the cells and blood on the surface will readily dissolve in your lysis buffer and you will get great DNA, with the feather material still intact.
What do you mean you don't get any results? You don't get enough DNA to see on gel or measure spectroPhotometrically? That's not important, even if you can't see or measure it, just use it in a PCR and see what you can amplify. If your PCR didn't work, yes you have to improve your extraction method.
If the molted feathers is the best material you have, need a better DNA isolation method. You can do a pretty good job digesting the keratin by using DTT that slowly breaks the SS crosslinks between the keratin monomers, so you can get to the additional (low amount of) DNA present inside the feather (with prolonged incubation at elevated temp). The finer you can cut/grind your feather material, the faster the dissolving will be.
Check this paper, it has a protocol for DNA isolation from old and molted feathers:
http://rspb.royalsocietypublishing.org/content/276/1672/3395.full
Goodluck
Thank you very much for the detailed answers. Much appreciated. What I mean by "no results" is was like you said, I couldn't visualise DNA on gel. I guess I will run the PCR and see if it works first.
Hi Burak,
Why don't you try NAI based method. We recently developed protocol for DNA extraction from F 98 cancer cells. It is working very well for us. We got problem with HeLa cells but we solved it with addition of SDS. We published protocols last year 2012 and this year. find the details below, go in supplemental information.
1) dx.doi.org/10.1021/ja306810w | J. Am. Chem. Soc. 2012, 134, 17366−17368
2) dx.doi.org/10.1021/tx400158g | Chem. Res. Toxicol. 2013, 26, 1031−1033
Guru
Thank you, I cannot access your publications at the moment. I will definitely check them out as soon as I am back at the university. And great work by the way.
You could try a 0.5 mL Microcon DNA Fast Flow or Microcon DNA Fast Flow PCR Grade filter unit (EMD Millipore Cat. MRCFOR100 or MRCF0R100ET) after PCI treatment to buffer exchange and concentrate prior to PCR. No need for ethanol ppt. step.
Here is another reference that may be useful to you:
Rudnick, J.A., T.E. Katzner, E.A. Bragin, and J.A. DeWoody. 2007. Species
identification of birds through genetic analysis of naturally shed feathers.
Molecular Ecology Notes 7: 757-762.
Thank you for your answers.
Dear Brian, that paper doesn't seem to include any information about the extraction phase. Thank you anyway. I am trying to find DTT and try it in my lysis step. I will provide you with further information after I test it.
What about pounding in liquid nitrogen? Put a mortar into a styrofoam box filled with liquid nitrogen. Put your sample into the mortar and pound it with a pestle. Then pour the powder into Dounce homogenizer and lyse with your lysis buffer. I would also use lysis buffer with guanidinium thiocyanate instead the buffer you use. I never extracted DNA from feathers but I think that could help.
Here are 2 articles, wich possibly will help you
http://www.rmrs.nau.edu/publications/BayardDeVolo_etal_2008/BayardDeVolo_etal_2008.pdf
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