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There are two major benefits for this change in protocol. One benefit is that it provides the option for starting with as little as 1 µg of total RNA using just the standard One-Cycle Labeling protocol. The other benefit is that it incorporates a more convenient and efficient experimental workflow.
Previously, IVT was carried out with 4-5 hours of incubation on the second day of a standard One-Cycle Target Labeling protocol. This made the procedure on the second day rather hectic, since users typically performed the cRNA cleanup (~30 minutes) and fragmentation (~1 hour) after the IVT in order to hybridize their samples on the arrays overnight.
In order to make the workflow more flexible, the new GeneChip IVT Labeling Kit provides a general guideline of using an overnight incubation. However, for customers who prefer the previous protocol, they have the option to spike in additional T7 RNA Polymerase (Ambion, P/N 2085, as described in the product insert) to reduce the IVT reaction time to 4-5 hours.
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