Recommended DNA Extraction methods for ONT Sequencing

1) Sambrook and Russell DNA extraction.

This protocol was modified from Chapter 6 protocol 1 of Sambrook and Russell51. 5 × 107 cells were spun at 4500g for 10 min to pellet. The cells were resuspended by pipette mixing in 100 μl PBS. 10 ml TLB was added (10 mM Tris-Cl pH 8.0, 25 mM EDTA pH 8.0, 0.5% (w/v) SDS, 20 μg/ml Qiagen RNase A), vortexed at full speed for 5 s and incubated at 37 °C for 1 h. 50 μl Proteinase K (Qiagen) was added and mixed by slow inversion ten times followed by 3 h at 50 °C with gentle mixing every 1 h. The lysate was phenol-purified using 10 ml buffer saturated phenol using phase-lock gel falcon tubes, followed by phenol:chloroform (1:1). The DNA was precipitated by the addition of 4 ml 5 M ammonium acetate and 30 ml ice-cold ethanol. DNA was recovered with a glass hook followed by washing twice in 70% ethanol. After spinning down at 10,000g, ethanol was removed followed by 10 min drying at 40 °C. 150 μl EB (Elution Buffer) was added to the DNA and left at 4 °C overnight to resuspend.

2) Qiagen Gentra® Puregene® kit (100-200 kb)


3) Plant gDNA extraction for long read seq

Five grams of flash frozen leaf tissue was ground in liquid nitrogen and extracted with 20 mL CTAB/Carlson lysis buffer (100 mM Tris-HCl, 2% CTAB, 1.4 M NaCl, 20 mM EDTA, pH 8.0) containing 20 μg/mL proteinase K for 20 min at 55 °C. The DNA was purified by addition of 0.5× volume chloroform, which was mixed by inversion and centrifuged for 30 min at 3000 RCF, and followed by a 1× volume 1:1 phenol: 24:1 chloroform:isoamyl alcohol extraction. The DNA was further purified by ethanol precipitation (1/10 volume 3 M sodium acetate pH 5.3, 2.5 volumes 100% ethanol) for 30 min on ice. The resulting pellet was washed with freshly prepared ice-cold 70% ethanol, dried, and resuspended in 350 μL 1× TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) with 5 μL RNase A (Qiagen, Hilden) at 37 °C for 30 min, followed by incubation at 4 °C overnight. The RNase A was removed by double extraction with 24:1 chloroform:isoamyl alcohol, centrifuging at 22,600×g for 20 min at 4 °C each time. An ethanol precipitation was performed as before for 3 h at 4 °C. The pellet was washed as before and resuspended overnight in 350 μL 1× TE.
Genomic DNA sample was further purified for ONT sequencing with the Zymo Genomic DNA Clean and Concentrator-10 column (Zymo Research, Irvine, CA).

4) The "Mayjonade" protocol


Created by patrig. Last Modification: 2018-11-06 16:00 by patrig.