We are trying to isolate DNA from liver tissue of snake preserved in alcohol/RNAlater using phenol:chloroform method. However, we are not getting good quality DNA. There is smearing or trailing of DNA. We did not use RNase. What may be the reason and how do we troubleshoot the problem?
20-30 mg tissue was chopped well into pieces and suspended in TE buffer. 4M GuHCl was added, followed by 20 ul Proteinase K. Incubated at 55C till tissue was lysed. 0.1-0.2% SDS was added and left for 15 min at RT. Phenol:Chloroform:Isoamyl was added and centrifuged at 12000 rpm for 10 min. The aqueous phase was taken and chloroform was added, centrifuged and aqueous phase was taken. The DNA was precipitated with 95-100% ethanol, centrifuged and the pellet was washed with 70% ethanol. The DNA was then suspended in water/TE buffer.
I think Rajeev you can isolate hepatocyte from liver tissue and then try to make DNA . This will help you to make good quality DNA
Samples were stored either in RNAlater or alcohol. The result was same for most of the samples, except some where the DNA quality was good enough. The DNA is to be used for PCR. We will skip GuHCl step and use SDS instead. Any other suggestion?
First perhaps phenol-chloroform is to aggressive. You can do it with a wonderful methods I show my pupils, I send you the protocol in the 3th of january. In it you can obtain at the end, liquid with the DNA clean and nice. The good thing is you need to fish the DNA with a little hook and translate to another eppendorf. Children are happy to see the double DNA with their property eyes.
and of course you need RNase to destroy RNA if you want to use phenol chloroforms method
And remember liver have a lot of aggressive substances who can destroy the DNA. In snake a lot of toxic substances there are and you need to go to Methods in Enzymology to obtain a protocol good enough for tissue with special difficulty properties. Also you can obtain first the nucleus of the material and after when you have clean nucleus you can obtain from them the dna
But the methods for students is more wonderful, is like do a dance or a good film, they and you feel it is a nice day, I see at last my DNA. You now if you are working you need to change things everyday to do the protocol correctly but to add a little bit art in your live. ja, ja. See you in the next frontier of science and artist people.
If you continue with problems, there is a wonderful friend here in Valencia working with snakes and DNA from years and he is one of the best in the world working with venous of snake. Tell me if you are interested and I can give you the email of the lab of him
If you have good result with some samples...that means your protocol is ok with those samples. You should track down of what is the difference between the work/not work samples. Were they done by different person, or any differences in the "chopping" method, is it related to the time the samples were kept before extraction, How they clean up the equipments, etc.? DNase contamination during processing may be one of the suspect.
I've noticed that you have quite a bit of RNA on the gel. To see the problem clearly, if there is problem at all, my suggestion is first to get rid of the RNA with RNase, clean up with alcohol ppt, then measure the OD and run gel at the quantity visible on the gel (ca. 20 ng/well should be suffice) to inspect for the quality. Good luck.
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