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strange transcription with T7 RNA polymerase

in-vitro transcription

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#1 lo_ken

lo_ken

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Posted 12 August 2013 - 01:59 PM

Hi all,

 

I am encountering a very strange problem that I do not understand as I am relatively new to in-vitro transcription, so I am hoping to leverage others who do this a lot.

 

Here is my problem.  I am trying to express 5 small RNAs (25 to 65bp) and perform some downstream reactions with them.  So I designed oligonucleotides that contains the T7 promoter upstream of my desired complementary DNA sequences (5 different oligos).  I mix the 5 different oligos at the same molar concentration and perform a 10-fold dilution series.  I then add the T7 RNA polymerase complement (another oligo) and perform hybridization by heating to 60C then ramping temperature down at 0.1C /s to RT.  Then I add T7 RNA polymerase and incubate at 37C for 3 hours.  I then run a denaturing PAGE on the product.

 

What is strange is that I see a laddering pattern at ~20, 40, 60, 80, 100, 120, 140, 160, 200, ... all the way past 300bp (I can tell too well beyond 300 'cos I am running a 15% TBU gel) for all of the reactions.  This is especially apparent in the lower concentrations (I have one where I didn't put in any template and I see the exact same thing).  I then tried to exclude the T7 RNA polymerase complement and the lane turns up empty.  

 

I also tried digesting the in-vitro transcription product with RQ1 (RNase free DNase) and the laddering remains, meaning that the product is indeed RNA.

 

I switched vendors and reproduced the exact same pattern (I started with the Ambion in-vitro transcription kit and then switched to Promega's RiboMAX in-vitro transcription kit).  Both produced the laddering pattern.

 

I tried looking through the literature to see if anyone has seen this before, but no luck so far.  Perhaps someone more experienced with this would be able to point to something trivial.  Please help.

 

I am attaching a gel picture of the phenomenon.

 

Best,

 

Kentoolroom 2013-08-09 15hr 27min_markup.jpg

 






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