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Genomic DNA contamination in RNA sample


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#1 marg1

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Posted 29 April 2009 - 08:08 AM

Hi!
I have some problems to perform a RT-PCR becouse I have in my RNA sample a genomic DNA contamination. I treated my RNA sample using different DNase enzyme, but it didn't work. And I cannot using PCR primers construct in two exons.
Do you have any suggestion about it?
Thanks in advance

#2 bob1

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Posted 29 April 2009 - 04:30 PM

Unfortunately there isn't much you can do about it if you can't make exon spanning primers and the DNase doesn't work. I would have another go with the DNase, making sure it is fresh as they aren't very stable. I would also be tempted to do another RNA extraction being very very careful to not take over any of the interphase layer if doing Trizol or equivalent extractions.

#3 dvddecarvalho

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Posted 01 May 2009 - 04:19 PM

As bob1 sad DNAse is very unstable. Make sure to not vortex it after melting as DNAse is mechanicaly sensitive. Mix it gently.
Another thing you could do is to dilute more your cDNA sample. Sometimes dilutions of 500x or 1000x are fine to avoid DNA amplification.

Good look,
Dvd

Edited by dvddecarvalho, 01 May 2009 - 04:29 PM.





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