qiime_experiment_820:639907:9
The extracted DNA was not pooled, and each sample represents the DNA from the biofilm of single kelp individual. DNA was eluted with 30 ul water and samples were diluted accordingly to a final concentration of 20 ng/L. DNA samples were quantified using a Nanodrop spectrophotometer (Nyxor Biotech, Paris, France).
Multiplexed bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) was performed using the Titanium platform (Roche Applied Science, Indianapolis, IN) as previously described (Dowd et al. 2008) in a commercial facility (Research and Testing Laboratories, Lubbock, TX). Briefly, a single step PCR using the primers that span the variable regions V1???V3 of the 16S gene, 28F 5??_GAGTTTGATCNTGGCTCAG and 519r 5??_GTNTTACNGCGGCKGCTG), was used to amplify the 16S rRNA genes as well as to introduce adaptor sequences and sample-specific 8-mer oligonucleotide tags (barcodes) into the DNA. The same bar codes were utilized for each primer set. The initial generation of the sequencing library used a one-step polymerase chain reaction (PCR) with a total of 30 cycles, a mixture of HotStart and HotStar high fidelity taq polymerases (Qiagen).
experiment_title
Kelp associated bacteria
key_seq
TCAG
sample_center
MBRI
sequencing_meth
pyrosequencing
run_date
6/7/10
experiment_center
MBRI
pcr_primers
FWD:GAGTTTGATCNTGGCTCAG; REV:GTNTTACNGCGGCKGCTG
region
2
run_center
MBRI
target_subfragment
V13
platform
Titanium
library_construction_protocol
Multiplexed bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) was performed using the Titanium platform (Roche Applied Science, Indianapolis, IN) as previously described (Dowd et al. 2008) in a commercial facility (Research and Testing Laboratories, Lubbock, TX). Briefly, a single step PCR using the primers that span the variable regions V1???V3 of the 16S gene, 28F 5??_GAGTTTGATCNTGGCTCAG and 519r 5??_GTNTTACNGCGGCKGCTG), was used to amplify the 16S rRNA genes as well as to introduce adaptor sequences and sample-specific 8-mer oligonucleotide tags (barcodes) into the DNA. The same bar codes were utilized for each primer set. The initial generation of the sequencing library used a one-step polymerase chain reaction (PCR) with a total of 30 cycles, a mixture of HotStart and HotStar high fidelity taq polymerases (Qiagen).
experiment_design_description
The extracted DNA was not pooled, and each sample represents the DNA from the biofilm of single kelp individual. DNA was eluted with 30 ul water and samples were diluted accordingly to a final concentration of 20 ng/L. DNA samples were quantified using a Nanodrop spectrophotometer (Nyxor Biotech, Paris, France).
row_number
9
target_gene
16S rRNA
study_center
Stanford University