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Problem with in vitro transcription


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#1 Nancybio

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Posted 12 June 2014 - 05:08 PM

I have the viral gene inserted in the pGEM plasmid (done by Genscript). I am trying to set up the standard curve for the real time PCR detection of the virus. So, I cut the plasmid with SAC 1 to linearize it and then I gel extracted the linear plasmid. This linear plasmid I used as template for in vitro transcription with T7 ribomax kit. And at this step I am stuck. The control of the kit did not work, so I got new kit. Then the plasmid gave me multiple RNA bands. Then I also used Klenow but there was no difference when I used the Klenow. Now again, the kit does not work with either the control or the sample. I am following all the good laboratory practices and still I have lot of problems for 2 months with this kit. Can anyone suggest me some tips or a better kit. Thanks!

 

- really frustrated !!

 



#2 Ahrenhase

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Posted 13 June 2014 - 12:25 AM

I use ambions Mmessage (am1344) for IVT of my pGEM vectors.  I cut with Spe1 because I have an ACTAGT after my polyA sequence, but your construct may differ.

 

My protocol involves digesting the plasmid overnight, DNA cleanup (no gel extraction), run mmessage kit (1ug template per reaction).  This generally yields me 20x mRNA per 1x input template DNA.  When I run out on a nondenaturing gel, I see my band of interest but sometimes also see a higher molecular weight band.  Due to the nature of my experiment, I don't think too much of this higher molecular weight band, but I should probably investigate what it is... 






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