Hello every one i need help i wana make dsRNA using Ribomax large Scale RNA Production system to use T7 promoter primer to extract DNA from PCR product but i cant understand protocol provide by manufacturars its very technical? i need to inject drRNA to insect to knokdown gene if some body do the same experiment can you please help me to send me simple protocol step wise thanks.
An off target minimized approximately 300 or 500bp gene region were identified
employing online software (ex. dsCheck http://dscheck.rnai.jp/). The T7 promoter
sequences were added to the 5' end of the gene specific primers (selected region) which were used for dsRNA synthesis. DNA templates for dsRNA were synthesized using normal PCR (Gene isolate used as template DNA). The amplified PCR product was resolved on 1% agarose gel and eluted using gel extraction kit (Macherey Nagel,
Germany). Eluted products were quantified and 1 μg of the same was used for in vitro
transcription using NxGenR T7 RNA Polymerase (Thermo Scientific, Fermentas,
Lithuania) or any other kit and purified the dsRNA (Sambrook and Russel, 2001). dsRNA concentration was determined by Nanodrop spectrophotometer and its integrity should analysis on 1.5% agarose gel.
- Badges
- Science method