Hi all
I am having trouble getting a MiSeq run to work and was hoping someone might be able to help
I have swab samples from surfaces in a hospital, i have DNA extracted using a bead beat and Qiagen column. Then I have pooled the extracted DNA from bed spaces and PCR's using 16S primers and custom barcodes for my library prep.
I have qPCR'd almost all of the samples individually before pool with a Staph generic assay and there are a few with not much in and some that may have been inhibited but I would think that would be diluted out with the pooling.
So, I have PCR'd to add barcodes, run a size select gel to purify - seen bands on all lanes. I have Qubited, adjusted to 4nM, pooked 96 samples, qubited again and then used this as my library. Denatured and prepared as per Illumina protocol and the samples are not working.
Does anyone have any ideas? thanks
I am having trouble getting a MiSeq run to work and was hoping someone might be able to help
I have swab samples from surfaces in a hospital, i have DNA extracted using a bead beat and Qiagen column. Then I have pooled the extracted DNA from bed spaces and PCR's using 16S primers and custom barcodes for my library prep.
I have qPCR'd almost all of the samples individually before pool with a Staph generic assay and there are a few with not much in and some that may have been inhibited but I would think that would be diluted out with the pooling.
So, I have PCR'd to add barcodes, run a size select gel to purify - seen bands on all lanes. I have Qubited, adjusted to 4nM, pooked 96 samples, qubited again and then used this as my library. Denatured and prepared as per Illumina protocol and the samples are not working.
Does anyone have any ideas? thanks
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