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|total DNA was liberated from cells by heating their suspension in water for 5 minutes at 95°C and removing cellular debris by spinning at 13200×g for 3 minutes at room temperature. For total RNA preparation, cells were pulverized in liquid nitrogen and RNA was isolated using TRI Reagent (Sigma) followed by purification with NucleoSpin RNA Clean-up (Macherey-Nagel). For total cDNA preparation, 2 µg of total RNA was used with RevertAid M-MuLV Reverse Transcriptase (Fermentas) according to the manufacturer’s recommended protocol.
|The following might help to increase RNA yield
- Heat the DEPC Water to 70°C before adding to the column.
- Increase the incubation time to 5 minutes.
- Increase the elution volume.
- Repeat the elution step with fresh DEPC Water (this may increase the yield, but decrease the concentration).
- Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).
|Isolated RNA was treated with RNase-free DNase I
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