Semi-Q-RT-PCR

"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 manufacturer’s 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 µl), forward primer and reverse primer (200nM), RNase inhibitor (5 units) and remaining volume was made up with DEPC treated water to 25 µl. The thermal cycler was programmedas follows: initial reverse transcription at 42ºC for 30 min, RT enzyme enaturation at 95ºC for 10 min followed by 30-35 cycles of cDNA amplification. OMICS Group International is one of the leading Open Access Publishers which is publishing 700+ peer-reviewed journals with the support of 50,000+ editorial board members as editorial team and aimed to disseminate the scholarly knowledge to the scientific society. OMICS Group also organizing 3000+ International Scientific Conferences and events yearly all over the world with the support of 1000+ Scientific associations worldwide.

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Last date updated on April, 2024