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I am planning to deep sequence material from 13 samples. For this, I am isolating RNA from various organs, reverse transcribing with a gene specific primer and performing a multiplex PCR (~400 bp fragments). Then, I will ligate a barcode to the PCR product from each sample and sequence. Since I am jumping into the middle of the standard 454 prep, can someone please tell me how much starting material I need for each sample for a full 454 plate?
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Hi Hambone,
Internally, the development team has been using PCR products with ~100 to 500 ng of material. They are starting with the End Repair step of the 454 Rapid Library Preparation protocol and with good success.
I hope this helps,
Nicole
Technical Support Scientist
454 Life Sciences, A Roche Company
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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