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RT-PCR

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In advance prepare master mixes in RNase-free 0.5 ml tubes consisting of :

  1. 2ul dNTP mix (Pharmacia, 25 mM each dNTP)
  2. 2ul Oligo-dT primer (200 pMol)
  3. 5ul Super RT buffer
  4. 26.5ul DEPC-treated water

Store them on ice.
The RNA (10ul containing ~1.5ug mRNA) was added to the tube and mixed by carefully pipetting up and down. The mixture was overlaid with one drop of mineral oil (Sigma) and heated to 67℃ for 5 minutes in a thermocycler (Biometra, Trio Thermoblock) to disrupt secondary structures. The mixture was then cooled down to 42℃ and 2ul RNAsin (Promega) and 2.5ul Super RT (HT Biotechnology Ltd, Cambridge, UK) were added. The reaction was incubated for 1 hour at 42℃ and then heated to 100℃ for 3 minutes (after the lid was punctured with a sterile needle). The single-stranded cDNA containing reaction was briefly centrifuged and stored at -20℃


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