Berglund:Transcription: Various Methods

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Before attempting to do transcription for the first time, I (Amy) highly recommend that you read these two papers:

J.F. Milligan and O.C. Uhlenbeck. 1979. Synthesis of small RNAs using T7 RNA polymerase. Advanced Cyclic Nucleotide research. 10, 135 (pgs 51-62).

J.F. Milligan, D.R. Groeve, G.W. Witherell, O.C. Uhlenbeck. 1987. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. 15, 21 (pgs 8785-8798).

Read the notes below in order to optimize whatever type of transcription you are attempting.

Be aware. What is key in transcription is quality. Spermidine, once diluted into water, is not stable for longer than a year. You will see the quantity of your transcription product decrease as spermidine breaks down. The T7 polymerase can last a long time once stored in the -20, but it is always good to never assume that it lasts forever. Nucleotides MUST be pHed to 8.1 or you will see a significant decrease in transcription product. Ribonucleotides are not stable forever, making new stocks is a good idea every 4-5 months in order to keep transcription quantities optimal.

Be aware. pH of the transcription reaction is key, you must be around pH8 AT THE TEMPERATURE you are transcribing at. pH of certain buffers is temperature dependent.

Some templates benefit from a short transcription time ie 1-2 hours at 37°C. Other templates (sometimes longer ones) do better if you go overnight at room temperature.

It's best to add Rnasin to your transcriptions.

Notes

Materials

classic method

Hot, Capped RNA Method 1

Hot, Capped RNA Method 2

Large Scale: 10ml

From oligomers

cold SHAPE method(Amy)

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