DNA preparation: 5ug of Axolotl gDNA was fragmented to 10kb using the HydroShear. Following end repair and dA tailing, Not1S adapter was ligated. Ligated DNA was amplified for 10cycles using LongAmp taq. Avg size following amplification approx. 5kb. Amplified DNA used for ONT library prep according to the new SEQMAP004 protocol using his-tagged beads. Protocol is as follows: ~ 2ug of amplified DNA was end repaired and purified using ampure beads using 0.7:1 ratio. End repaired DNA was dA tailed then taken forward for ligation of ONT adapter. Following ligation purification of adapter ligated DNA was carried out using his tagged beads. Total recovery ~75ng.
dna extraction protocol
DNA was prepared from red bood cells as follows.Blood cells were resuspended in 1x SET buffer and spun for 3 minutes at room temperature. The pellet was resuspended in 1X SET buffer,proteinase K (200ug/ml) and SDS (0.5%). Cells were incubated overnight at room temperature. Genomic DNA was phenol:chloroform purified twice and precipitated. DNA was removed by spooling onto a glass rod. The DNA was resuspended in TE buffer over 2 days at room temperature with gentle rocking.
kit
SQK-MAP004
sequencing protocol
gDNA