My problem is that I obtained 18s hostgene expression  appears on very late cycles. cDNA quality may be low. OD260/280:1.68 and 640ng/ul

500ng RNA+ NF water to final volum 10ul is warmed 70 C for 5 min,

Then added  10ul Mastermix to RNA (total volum 20ul) and incubate it 42 C for 1 hour and 94 C 2 minute andd 4 C forewer.  

Mastermix:

  • AMV buffer :4ul/reaction
  • DNTPs : 2ul
  • Random primers 1ul
  • Water: 1ul
  • RNAsin: 1ul
  • RTase: 1ul                
  • Finally 40 ul water is added to tubes. cDNA ready.

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