Imran Mungrue imran.mungrue@utoronto.ca

RESUME

METHODS

5) RT-PCR

*Incubate RNA with RNAse free DNAse1 at room temp for 15min.      0-5ug RNA      1ul DNAse1 (126U/ul)      2ul 10X DNAse Buffer      Sterile water      -------------------      20ul total volume *Add 2ul 25mM EDTA *Heat kill at 65degC for 10min RT Reaction *Anneal hexamers to RNA     22ul above      1ul Random Hexamers (100uM)     ---------------------      23ul total volume *Incubate at 65degC for 10min., chill on ice. *Leave at room temp for 10 min. to allow hexamer binding. (*Remove 2-4ul aliquot for RT- control, do not add RTase) *Add to each sample      8ul 5X 1st Strand Buffer      4ul 0.1M DTT      1ul 10mM dNTP      1ul 40U/ul RNAse Inhibitor *Pre-incubate at 42degC for 2min. *Add 2ul Superscript (2000U/ul) *Incubate at 42degC for 50min. *Heat kill at 75degC for 15 min. *Use 2ul to proceed with PCR as follows.      cDNA template     2ul      10X PCR Buffer   2.5      10mM dNTP        0.5      25uM pF              0.5      25uM pR              0.5      Taq Pol (5U/ul)     0.1      Sterile water        18.9