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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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