in vitro transcription

brett brett at BORCIM.WUSTL.EDU
Mon Sep 28 09:09:07 EST 1998


>Hello
>
>I am working with in vitro transcription and I have some problems with
>getting rid of the template DNA after the in vitro transcription. In the
>protocol they recommend to treat the sample (100 ul) for 10 min. with 5U
>RNase free DNase. I did that and I even tried to treat the sample for 2
>hours and there were still DNA left. (analyzed by running PCR on the
>sample without adding reverse transcriptase). I have heard that some DNA
>polymerases have RT activity, but since I haven't observed this
>phenomenon in any of my previous reaction where I use the same DNA
>polymerase I find it hard to believe that this can be the explanation. I
>use 5 ug of DNA template which is a lot, but that is what they recommend
>in the protocol. I will be very pleased if anyone can help me with my
>problem.
>
>Marie Klinge Termansen
>IMMI, University of Copenhagen

This is  not too surprising.  5 ug of  template is a lot (you didn't mention
size, but my guess is >10e13 DNA molecules).  Given the sensitivity of PCR
(theoretically and in well designed reactions, „1 molecule), you're really
asking your DNase to do quite a lot.  Whenever this topic came up in this
forum in the past, someone would always claim they could quantitatively remove
their input template using a single round of DNase, leading me to just chuckle
because they never mention how sensitive their RT-PCR is.  In our lab, someone
who is using a RT-PCR sensitive down to ~100 molecules of input RNA (and ~10
molecules of input DNA) has actually done this troubleshooting and found it
necessary to use two rounds of digestion using a cocktail of DNases to remove
DNA below the limit of detection.  She plans to publish this method, so I won't
elaborate.  However, for this discussion to go forward, I suggest we start
dealing with this topic in a more quantitative manner.  Also, Marie, depending
on your intended use of the RNA, you could throw in a gel purification step
in addition to DNase treatment.


Brett Lindenbach
brett at borcim.wustl.edu
Dept of Molecular Microbiology
Washington University School of Medicine

"I have never let my schooling interfere with my education. "
     - Mark Twain





More information about the Methods mailing list