Preparation of DNA Template
- The DNA fragment was generated using specific primers for the gene of interest and made from a cDNA library.
- The fragment was digested with BamH1 and cloned into pBluescript II vector.
- The plasmid was linearized with restriction enzymes (New England Biolabs) and the antisense and the sense strand made by T3 RNA polymerase and T7 polymerase.
- The restriction enzyme digests were performed overnight at 37 oC in 20µl total volume containing 2µg DNA with 5 Units of restriction enzyme.
- The fragments were seperated on a 1.4% agarose gel and extracted using a Qiaex II gel extraction kit (Qiagen) under Rnase free conditions.
- The DNA template was then made up to 100µl and an equal volume of chloroform : isoamyl alcochol (24:1) and mixed.
- The aqueous layer was removed and the DNA precipitated with 1/10 volume of 3M Na Acetate and 2.5 volumes of ethanol.
- Incubate for 15 minutes at 4 oC, then spin for 10 minutes at 10,000g and wash the pellet with 70% ethanol.
- The pellet was resuspended in 20µl diethyl pyrocarbonate (DEPC, Sigma) treated water (H 20-DEPC).
In Vivo Transcription
- The transcription reactions were performed in 20µl volumes.
- Using the following reaction mixture made at RT:
- 10µl DNA fragment
- 2µl 10x transcription buffer
- 2µl 10x DIG RNA labelling mix (Roche)
- 0.5µl Rnase inhibitor (Roche)
- 1µl RNA polymerase (T3 or T7) (Roche)
- 4.5µl H 2O-DEPC
- The reaction was performed overnight at 37 oC.
- Stopped by the addition of 2µl 0.2M EDTA/H 2O-DEPC pH 8.0.
- The DIG labelled cRNA was precipitated with 1/10 volume 4M LiCl and 2.5 volumes of ethanol at –80 oC for 1 hour.
- The cRNA was recovered
- centrifugation at 10,000g for 15 min at 4 oC
- washed in 70% ethanol
- allowed to dry
- resuspended in 50µl H 2O-DEPC.