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 37oC
in 20uL total volume containing 2ug DNA with 5 Units of restriction
- The fragments were seperated on a 1.4% agarose gel and
extracted using a Qiaex II gel extraction kit (Qiagen) under Rnase free
- The DNA template was then made up to 100uL 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 20ul diethyl pyrocarbonate
(DEPC, Sigma) treated water (H 20-DEPC).
In Vivo Transcription
- The transcription reactions were performed in 20uL volumes.
- Using the following reaction mixture made at RT:
- 10uL DNA fragment
- 2uL 10x transcription buffer
- 2uL 10x DIG RNA labelling mix (Roche)
- 0.5uL Rnase inhibitor (Roche)
- 1uL RNA polymerase (T3 or T7) (Roche)
- 4.5uL H 2O-DEPC
- The reaction was performed overnight at 37oC.
- Stopped by the addition of 2uL 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 50uL H 2O-DEPC.