"Semi-quantitative RT-PCR was performed to check transcripts of the GFP transgene in fungal transformants. Prior to RT-PCR, RNA was isolated using Trizol reagent (Invitrogen, USA) and treated with DNase (Taurus scientific) as per the manufacturerâs protocol. DNaseI treated RNA was quantified using Nanodrop (Thermoscientifc, USA) and total RNA at the concentration of 500 ng was used as the template for RTPCR.One step RT-PCR kit (Bioscript) was used for this purpose. RNA template was mixed with reaction mix consisting of 1x RT-PCR buffer, one step enzyme mix (1 Âµl), forward primer and reverse primer (200nM), RNase inhibitor (5 units) and remaining volume was made up with DEPC treated water to 25 Âµl. The thermal cycler was programmedas follows: initial reverse transcription at 42ÂºC for 30 min, RT enzyme enaturation at 95ÂºC for 10 min followed by 30-35 cycles of cDNA amplification.
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Last date updated on July, 2014