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Help!What's wrong with the endonuclease digestion of total DNA of

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#1 swm



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Posted 08 March 2005 - 07:42 PM

Total DNA from activated sludge was extracted and purified to amplify the fragment of 16SrRNA by using eubacterial universal primers. Some of PCR products were directly using the HaeIII to produce RFLP patterns.Unfortunately,by trying different systems and ways of endonuclease digestion,I can not obtain any clear and distinct DNA patterns, What I get is faint patterns
and are almost invisible. The digestion system is PCR product 8ul,endonuclease HaeIII 2U,PCR buffer 1ul.However, using this system in bacterial isolates,I can get a clear pattern.What's wrong with my endonuclease digestion, I attemp different reaction system and electrophoresis conditions,what I get is the faint,unclear DNA pattern,which I post in attachment.
Could anybody can do me a favor and tell me what's the matter and how to solve this problem! Thanks a lot, I am so eager to your solution.

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