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In Vitro Transcription/Translation

1. Linearize plasmid with appropriate enzyme.
   
   
2. Run in agarose gel to check completion of digestion.
   
   
3. Phenol/Chlorophorm extract, ethanol precipitate.
   
   
4. Dissolve DNA pellet in TE at a concentration of 2 mg/20 ml.
   
   
5. Set up in vitro transcription (From Strategene Kit):
   
   
6. 20 ml 5X Transcription Buffer
   1.33 ml 0.75 M DTT
   2.5 ml RNAsin (Promega)
   20 ml 2.5 mM XTP (5 ml of each)
   20 ml (2 mg) Linearized DNA Template
   1 ml (10-20 U) T7 RNA Polymerase
   35.17 ml H₂O (To 150 ml) 
   
   Incubate 1 hr 37°C.
   Phenol/Chloroform X 1
   EtoH ppt. Chloroform/IAA X 1
   Optional: Run 2% of translation product on a minigel to check for the presence of RNA.
   
   
7. Redissolve the RNA pellet in 24 ml H₂O (Not TE).
   Aliquot to 1 ml per tube.
   
   
8. In vitro translation:
   35 ml Reticulocyte Lysate
   1 ml RNAsin
   1 ml 1 mM Amino Acid Mixture (Minus Methionine)
   1 ml RNA in H₂O
   5 ml 35S Methionine
   7 ml H₂O to 50 ml
   
   Incubate at 30°C 1 hr.

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