Q RT-PCR

1/27/04

 

Part 2: cDNA preparation

 

1.     Spec RNA (2uL in 100). Make sure to use filter tips and try not to contaminate with RNAses. Keep RNA on ice.

 

  1. Put 5ug RNA in fresh Eppendorf tube, RNAse free.

 

  1. Add 1/10V 3M NaOAc pH5.2  (RNAse free) and 2V 100% EtOH (RNAse free).

 

  1. Keep 30Õ at –80C (bury tube in the frost) and spin 30Õ at 4C.

 

  1. Pour off supe. Wash 1x 500uL 70% EtOH. Spin 5Õ RT.

 

  1. Air dry pellet. Resuspend RNA in 8uL DEPC water.

 

  1. Follow Superscript protocol (Invitrogen 11904-018).

 

  1. Mix 5ug RNA with 1uL random hexamers (50ng/ul) and 1uL 10mM dNTP mix.

 

  1. 65C for 5Õ, on ice 1Õ.

 

  1.  Make master mix 10X RT buffer (2uL/rxn), 25mM MgCL2 (4uL), 0.1M DTT (2uL), and RNAse out (1uL). Aliquot 9uL/rxn in PCR tubes adding to 10uL RNA/primer mix.

 

  1. 25C fof 2Õ.

 

  1. Add 1uL superscript II RT to each tube, mix and 25C for 10Õ. For one control tube, do not add the RT.

 

  1. 42C for 50Õ.

 

  1. 70C for 15Õ to terminate reaction.

 

  1. Add 1uL RNAseH and incubate for 20Õ at 37C. Store cDNA at –20C.