Can't get rid of RNA in my phage extraction. - (Aug/14/2005 )

Hi,
I have problems removing RNAs from my extracts. I keep on getting RNA contamination one extraction after another. Trust me, I have used a lot of RNase A (2.5mg/ml) but still not able to get rid of the RNAs. I added the RNase with incubation at 37 degree celcius for more than one hour. I kept my RNase A in TE + NaCl buffer at -20 degree celcius. People told me that the phenol chloroform that I am using is not good already because it gave a pinkish color during mixing and extracting. Will phenol chloroform extract and seperate DNA and RNA? If that is the case which part of the clear phase should I get? Is there any wrong with I am doing?

Thanks a lot for helping.

hi
check pH of phenol chlo mixture.
And it's better to do RNAse treatment in smaller but multiples volumes than in a big one.

Normally phenol will not remove RNA from DNA (acid phenol will remove DNA from RNA but this is not what you want). Remember that RNase A only digests ssRNA at certain nucleotide positions (I think between purines but I am not certain). This means that some RNA is resistant to RNase A digestion.

Two tips:

1. Use RNase T1 and RNase A together in your RNA digests as they cut at different sequences.

2. Do the RNase digestion at a higher temp to denature the RNA (~50C is good).

Daniel

Use less BigDye mix

Thank you so much for all the information, I will try to do the digest in 50C and get the pH of my phenol chloroform. By the way, what is the pH that I am suppose to see? and why?

Thanks a lot to Daniel and Fred, really appreciate it.

For DNA extractions you want your phenol to be alkaline (~pH 8).

Daniel

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