Experiment SRX300902 Download

TitleEnriched black molly reads
AliasEnriched black molly
Design descriptionHydrophobic protein A magnetic beads (NEB) (10 mg/ml) were suspended in bind/wash buffer (10 mM Tris pH 7.5, 1 mM EDTA, 300 mM NaCl, 1 % TritonX-100). To 1 ml of beads was added 100 µl of MBD-Fc protein and the sample mixed with rotation for 10 minutes at room temperature. The sample was placed on a magnetic rack for 2–5 minutes to concentrate beads on the inner wall of the tube. The supernatant was carefully removed with a pipette without disturbing the beads and the pellet washed with 1 ml of bind/wash buffer with rotation for 5 minutes. The sample was placed on the magnetic rack, the supernatant discarded, and the wash step was repeated. The beads were then suspended in 1 ml of bind/wash buffer for later use. The purified intact IMR 90 cell line, E. coli, human saliva, human blood, black molly fish and malaria DNA was mixed with MBD-Fc protein A beads in the following ratio: 160 µl of bead slurry for every 1 µg of input DNA mixture. For small scale reactions used in Ion Torrent sequencing, 80 µl of beads were used with 0.5 µg input DNA mixture. For construction of SOLiD 4 libraries using saliva DNA, 2 mls of beads were used with 12 µg input DNA. Similarly, 320 µl of beads were used with 2 µg of malaria/human DNA mixture in the Plasmodium study and 9.12 µg of DNA with 1.5 ml of beads in the black molly fish study. A homogenous slurry of Protein A magnetic beads was created by gently pipetting the mixture up and down, or alternatively by gently rotating the tube for 15 minutes. DNA mixture was added to the bead slurry, and the mixture incubated for 15 minutes at room temperature with rotation. Beads were separated by placing the tube on a magnetic rack for 5 minutes at room temperature, resulting in a tight cluster of the beads on the side of the tube. The supernatant was removed without disturbing the beads, and transferred to a clean microcentrifuge tube. The supernatant contained the prokaryotic DNA. Illumina library construction and sequencing (MiSeq, GAIIx): genomic DNA (500 ng) was sheared for using a Covaris S2 device to obtain an average fragment size of ~350 bp. Illumina paired-end sequencing libraries were constructed using the NEBNext® DNA library prep reagent set for Illumina (NEB #E6040) following the standard Illumina sample preparation protocol. PCR library amplifications were performed with an MJ Research Thermo Cycler PTC-225. Illumina PE 1.0 and 2.0 primers, or PE 1.0 and 2.0-derived indexing primers, were used to amplify adapter-ligated library fragments by PCR. Libraries were amplified using optimized PCR conditions described in [33].
Referencesno content
OrganismsPoecilia cf. sphenops 'black molly'
SubmitterNew England Biolabs
InstrumentIllumina MiSeq
Library nameno content
Library strategyWGA
Library sourceGENOMIC
Library selectionother
Library layoutSINGLE
Library construction protocolno content
Related objects
Linksno content