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Problem with DNase treatment of RNA for RT-PCR


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#1 PCRblues

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Posted 15 February 2005 - 07:15 PM

Hello all,

In my RT-PCR experiments, i am getting two bright bands consistently. I ran a control with-out RTransciptase and I got only one band. So i was happy thinking that this band in -RT control was from DNA contamination and the other one (which lies at the same region where i want it to be) is the 'correct' one. To get rid of that contaminated band, i treated the sample with DNAse, but now i am not seeing any band at all. Both the bands have disappeared. Did anyone face similar problem? Is there a chance that DNAse can actually destroy all the RNA in the sample? What could be the most possible reason for this observation? Any help is really appreciated.

thanks in advance.

#2 mortene

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Posted 04 March 2005 - 01:44 AM

Its probably contaminating DNA that is your problem. I will recommend the use of a new ds DNA specific DNAse (Shrimp DNase) that you can buy from USB in the states or ABgene in the UK. It does not cleave RNA or ssDNA and can be added to your RT mix. It will remove contaminating DNA during the RT step and can be easely be inactivated before PCR.

Good luck!




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