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
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 4oC, 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 (H20-DEPC).
In vitro 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) and 4.5µl H2O-DEPC the reaction was performed overnight at 37oC.
and stopped by the addition of 2µl 0.2M EDTA/H2O-DEPC pH 8.0. The DIG labelled
cRNA was precipitated with 1/10 volume 4M LiCl and 2.5 volumes of ethanol at –80oC
for 1 hour. The cRNA was recovered by centrifugation at 10,000g for 15 min at 4oC,
washed in 70% ethanol, allowed to dry and then resuspended in 50µl H2O-DEPC.