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T7 transcription- Scale up Problems

T7 transcription transcription T7

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#1 Toastayy

Toastayy

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Posted 22 June 2013 - 03:18 PM

Hey guys, we've been doing some Analytic Scale T7s (20ul) with a certain amount of DNA, lets say around 20ng, and have been getting pretty awesome results. From this we decided to scale up to produce enough RNA so we can do most of the experiments related to the project.

We scaled up 20 fold to 400ul T7 reactions, and used 20x more DNA template ~400ng. Unfortunately when we do this, the RNA yield we get on our RNA gels are very poor, and even worst after extraction and purification.

Can anyone point me in the general direction as to why this maybe happening and how to improve our RNA yield on the Prep scale?

We've been following (somewhat) the Sauer protocol, on T7 transcription and we've just hit a wall.

Thanks in advance.





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