Illustra genomiphi dna amplification kit

Seeds of CS-7EL were germinated, their meristem root-tip cells were synchronized and used to prepare suspensions of intact mitotic metaphase chromosomes [13 (link)]; GAA microsatellites on chromosomes were labelled by FITC [61 (link)] and chromosomal DNA was stained by DAPI. The samples were sorted in four independent batches using a FACSAria II SORP flow cytometer and sorter (Becton Dickinson Immunocytometry Systems, San José, USA); bivariate analysis GAA-FITC vs. DAPI was used to discriminate the population representing 7EL (Additional file 19). Chromosomal DNA was amplified individually using an Illustra GenomiPhi DNA amplification kit (GE Healthcare, Mississauga, Canada) following [62 (link)]. Four Truseq PCR-free (Illumina, San Diego, CA) paired-end libraries with varying insert sizes were prepared from a single pool of amplified DNA.

The genomic DNA of IOLA was extracted and amplified from a BALF specimen of patient KY-405 as outlined in Fig. 1. The BALF specimen (400 µL) was first filtered through a polytetrafluoroethylene (PTFE) filter with a 1.2-µm pore size (Sartorius Stedim) to remove host cells and larger bacterial cells. The filtrate was then passed through a filter with a 0.2-µm pore size (Advantec). After being washed twice with 1000 µL of PBS, the filter was transferred to a 15-mL tube and incubated with 2 mL of PBS containing 2% SDS at room temperature for 10 min. After phenol extraction (two times), the DNA in the aqueous phase was concentrated with an Amicon Ultra 0.5 mL 100 K filter (Merck Millipore) and amplified with an Illustra Genomiphi DNA Amplification Kit (GE Healthcare Life Sciences) according to the manufacturer’s instructions except in relation to the heat denaturation process. To selectively amplify the extremely AT-rich genomic DNA of IOLA, we optimized the denaturation conditions to 80 °C for 3 min. The amplified DNA was purified using the Wizard DNA Clean-Up System (Promega).